extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
6-Hydroxydopamine induces secretion of PARK7/DJ-1 via autophagy-based unconventional secretory pathway

ABSTRACT PARK7/DJ-1 is a Parkinson disease- and cancer-associated protein that functions as a multifunctional protein involved in gene transcription regulation and anti-oxidative defense. Although PARK7 lacks the secretory signal sequence, it is secreted and plays important physiological and pathophysiological roles. Whereas secretory proteins that lack the endoplasmic reticulum-targeting signal sequence are secreted from cells by way of what is called the unconventional secretion mechanism, the specific processes responsible for causing PARK7 to be secreted across the plasma membrane have remained unclear. In the present study, we found that PARK7 secretion was increased by treatment with 6-OHDA via the unconventional secretory pathway in human neuroblastoma SH-SY5Y cells and MEF cells. We also found that 6-OHDA-induced PARK7 secretion was suppressed in Atg5-, Atg9-, or Atg16l1-deficient MEF cells or ATG16L1 knockdown SH-SY5Y cells, indicating that the autophagy-based unconventional secretory pathway is involved in PARK7 secretion. We moreover observed that 6-OHDA-derived electrophilic quinone induced oxidative stress as indicated by a decrease in glutathione levels, and that this was suppressed by pretreatment with antioxidant NAC. We further found that NAC treatment suppressed autophagy and PARK7 secretion. We also observed that 6-OHDA-induced autophagy was associated with activation of AMPK and ULK1 via a pathway which was independent of MTOR. Collectively these results suggest that electrophilic 6-OHDA quinone enhances oxidative stress, and that this is followed by AMPK-ULK1 pathway activation and induction of secretory autophagy to produce unconventional secretion of PARK7. Abbreviations: 6-OHDA: 6-hydroxydopamine; AMPK: AMP-activated protein kinase; ATG: autophagy related; CAV1: caveolin 1; ER: endoplasmic reticulum; FN1: fibronectin 1; GSH: glutathione; IDE: insulin degrading enzyme; IL: interleukin; LDH: lactate dehydrogenase; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; NAC: N-acetyl-L-cysteine; PARK7/DJ-1: Parkinsonism associated deglycase; PD: Parkinson disease; RPS6KB1/p70S6K: ribosomal protein S6 kinase B1; RPN1: ribophorin I; ROS: reactive oxygen species; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type

Introduction
Most secretory proteins have a signal sequence at the N-terminus which allows them to be transported from the endoplasmic reticulum (ER) to the Golgi and exported from the cell via the conventional secretion mechanism. There are a smaller number of proteins that lack the ERtargeting signal sequence which are secreted from the cell by what is referred to as the unconventional secretion mechanism [1][2][3]. Unconventionally secreted proteins include DBI (diazepam binding inhibitor, acyl-CoA binding protein), IL1B (interleukin 1 beta), IL18 (interleukin 18), HMGB1 (high mobility group box 1), and FGF2 (fibroblast growth factor 2) [4][5][6][7]. Although it is known that these unconventionally secreted proteins are released from the cell via an ER-/Golgi-independent pathway, the specific mechanism by which the soluble protein is able to translocate across the hydrophobic plasma membrane still remains to be discussed. Multiple pathways have been proposed as possible routes by which such unconventional secretion might be accomplished, these proposed routes including secretory lysosomes, plasma membrane shedding, exosome derived from multivesicular bodies, and secretory autophagy [1][2][3]8]. Because some of the proteins that are secreted via the unconventional secretory pathway play important extracellular functional roles in developmental processes and inflammation, the unconventional secretion mechanism is being studied extensively.
PARK7/DJ-1 (Parkinsonism associated deglycase) was initially identified as an oncoprotein that cooperates with Ras to promote cell transformation [9]. PARK7 has been implicated as a protein encoded by one of the causative genes (PARK7) in a familial form of Parkinson disease (PD) [10]. It has also been suggested that PARK7 is related to a sporadic form of PD [11,12]. Elevated levels of an oxidized form of PARK7 (oxPARK7) are observed in erythrocytes of PD patients at early stages [11]. oxPARK7 is also observed in the brains of human PD patients and animal PD models [12]. PARK7 is a multifunctional protein involved in several molecular processes such as gene transcription regulation, protein stabilization, and anti-oxidative defense [13,14]. Overexpression of PARK7 can protect dopaminergic neurons against several types of oxidative stress by increasing cellular glutathione (GSH) levels [15]. Knockdown of PARK7 increases susceptibility to oxidative stress [15,16]. Human PARK7 has three cysteine residues at amino acid numbers 46, 53, and 106, and undergoes preferential oxidation at position 106 (Cys106) under oxidative stress [17,18]. Replacement of Cys106 with alanine, serine, or aspartic acid causes PARK7 to lose its anti-oxidative properties.
PARK7 is localized in the cytoplasm and the nucleus; it has also been shown that some PARK7 is localized in the mitochondria [9,10,19,20]. Several lines of evidence suggest that PARK7 is secreted from cells into the circulation despite the fact that PARK7 lacks a secretory signal sequence [21][22][23][24][25][26][27][28][29]. Increase in PARK7 secretion into serum or plasma has been observed in patients with breast cancer, melanoma, stroke, familial amyloid polyneuropathy, and PD [21][22][23][24][25][26][27]. There have been conflicting reports of both increase [28] and decrease [29] in the amount of PARK7 in cerebrospinal fluid in sporadic PD patients as compared with control groups. Secreted PARK7 plays important physiological and pathophysiological roles which include involvement in anti-oxidative effects [30], extracellular signaling between neighboring neuronal cells [31], angiogenic and osteogenic factors [32], and degradation of aggregated protein [26]. Such findings indicate that PARK7 secretion may be implicated in the etiology and/or progression of diseases such as PD and cancer, possibly making it suitable for use as a biomarker. Despite the considerable number of such findings, however, it remains unclear how cytosolic PARK7 protein is able to translocate across the plasma membrane.
In our present study, to investigate the molecular mechanism of PARK7 secretion, we use 6-hydroxydopamine (6-OHDA), which is a catecholaminergic neurotoxin commonly used to generate experimental models for PD in vivo and in vitro [33]. As a result of our study, we find that PARK7 secretion is increased by treatment with 6-OHDA via the unconventional secretory pathway in human neuroblastoma SH-SY5Y cells and mouse embryonic fibroblast (MEF) cells. We demonstrate that 6-OHDA induces oxidative stress as indicated by a decrease in GSH levels, resulting in induction of macroautophagy/autophagy. We further show that 6-OHDA-induced autophagy is associated with activation of AMP-activated protein kinase (AMPK) and its downstream effector ULK1 (unc-51 like autophagy activating kinase 1) and that this occurs via a pathway that is independent of MTOR (mechanistic target of rapamycin kinase). We conclude that AMPK-ULK1-mediated secretory autophagy plays an important role in the unconventional secretion of PARK7.

PARK7 is secreted under non-stress conditions in SH-SY5Y cells
To assess PARK7 secretion from human neuroblastoma SH-SY5Y cells, cells were cultured in serum-free medium for 0-6 h to prevent contamination by serum protein. As controls, FN1 (fibronectin 1) was used as a protein marker secreted via the conventional pathway and RPN1 (ribophorin I) was used as a cell resident protein. Our results showed that PARK7 was secreted in a time-dependent manner similar to the observed secretion of FN1 control, and that RPN1 was present only in the cell lysate fraction (Figure 1(A and (B)). Evaluation was carried out to determine whether LDH (lactate dehydrogenase), an enzyme normally found only in the cytoplasm, was being released from the cell under the conditions tested, as a result of which it was found based on the small amount of LDH released that the PARK7 secretion observed was not due to plasma membrane leakage (Figure 1(B)). To evaluate whether PARK7 secretion was mediated by the conventional ER-/Golgi-dependent secretion mechanism, cells were treated with brefeldin A, an inhibitor of ER-Golgi transport, as a result of which it was found that treatment with brefeldin A inhibited FN1 secretion but not PARK7 secretion ( Figure 1(C), suggesting that the conventional secretory pathway was not involved in PARK7 secretion. As previously reported [9,10], most of PARK7 was found to be in the cytosolic protein-enriched fraction obtained by subcellular fractionation (Figure 1(D)), supporting the idea that PARK7 was secreted via an ER-/Golgi-independent secretory pathway. We also used 2D-PAGE to examine the oxidative state of PARK7, as a result of which we found that the ratio of oxPARK7 to total PARK7 in medium was almost the same as that in cells, suggesting that secretion of PARK7 was not induced by its oxidation (Figure 1(E)).
Treatment with 6-OHDA enhances secretion of PARK7 from SH-SY5Y cells We then evaluated the effect of 6-OHDA on PARK7 secretion. Because we had noticed that 6-OHDA in medium interfered with protein precipitation during the trichloroacetic acid precipitation procedure, protein secretion was evaluated using the conditioned medium obtained following 6-OHDA treatment as described in Materials and Methods. Results showed that 6-OHDA treatment for 3 h increased PARK7 secretion in a concentration-dependent manner (Figure 2(A)). Significant release of LDH from 100 μM 6-OHDA-treated cells was not observed (Figure 2(B)), suggesting that the increase in PARK7 secretion was not the result of plasma membrane disruption by 6-OHDA. Brefeldin A treatment did not inhibit PARK7 secretion (Figure 2(C)), confirming that 6-OHDA-induced PARK7 secretion was mediated via an ER-/Golgi-independent secretory pathway. Because it had recently been reported that CASP1 (caspase 1) may act as regulator of unconventional protein secretion [34], we decided to assess the respective effects of a pan-caspase inhibitor (zVAD; i.e., Z-VAD [OMe]-FMK) and a specific CASP1 inhibitor (YVAD; i.e., Ac-YVAD-CMK) on PARK7 secretion. Our results indicated that 6-OHDA-induced PARK7 secretion was inhibited by either inhibitor (Figure 2(D)). Taken together, the foregoing series of results suggest that 6-OHDA induces PARK7 secretion via the unconventional secretory pathway.

6-OHDA-induced oxidative stress is implicated in PARK7 secretion
Considering the prominent effect of 6-OHDA on PARK7 secretion that we observed as reported above, we then sought to explore the mechanism of 6-OHDA-induced PARK7 secretion. It has been known that 6-OHDA is readily oxidized by molecular oxygen to produce superoxide anion, hydrogen peroxide (H 2 O 2 ), and 2-hydroxy-5-(2-aminoethyl)-1,4-benzoquinone (6-OHDA quinone), resulting in induction of intracellular oxidative stress [35]. We therefore decided to examine whether 6-OHDA-induced oxidative stress affected PARK7 secretion. We first assessed the effect of 6-OHDA on intracellular antioxidant GSH content, finding as a result that there was a significant decrease in GSH levels in cells treated with 100 μM 6-OHDA for 3 h (Figure 3(A)). We next examined whether the 6-OHDA-induced reduction in GSH level caused PARK7 secretion in the presence or absence of the GSH precursor N-acetyl-L-cysteine (NAC), a well-established cysteine supplier. We found that pretreatment with NAC suppressed the 6-OHDA-induced PARK7 secretion ( Figure 3(B)). Since we had previously reported that about 260 μM H 2 O 2 and 600 μM 6-OHDA quinone are formed from 600 µM 6-OHDA in medium [36], we decided to test the effect of H 2 O 2 on PARK7 secretion, finding as a result that treatment with 100 μM H 2 O 2 in the presence or absence of catalase did not increase PARK7 secretion (Figure 3(C)). Based on the foregoing series of results, we hypothesized that electrophilic quinone-induced oxidative stress might be responsible for 6-OHDA-induced PARK7 secretion. To test this possibility, we evaluated the effect of another electrophilic p-quinone, benzoquinone, on PARK7 secretion, finding as a result that, at non-cytotoxic concentrations (i.e., no release of LDH), benzoquinone induced PARK7 secretion in a concentration-dependent manner (Fig. S1A). Furthermore, NAC treatment was found to suppress the increase in 20 μM benzoquinone-induced PARK7 secretion (Fig. S1B). Similar to the effects of 6-OHDA treatment, 20 μM benzoquinone was found to reduce GSH content, and it was moreover found that this reduction could be abrogated by NAC treatment (Fig. S1C). Together, these results suggest that electrophilic quinone-induced oxidative stress underlies 6-OHDA-induced PARK7 secretion. We also examined whether PARK7 were quantified by densitometric scanning and the percentage of secreted PARK7/total PARK7 is shown. LDH release in the conditioned medium was analyzed by LDH assay. n = 3; mean ± S.D.; *, p < 0.05; **, p < 0.01. (C) SH-SY5Y cells were treated with 2 μg/ml brefeldin A in serum-free medium for 3 h. Whole cell lysates and the conditioned medium were immunoblotted with antibodies specific for PARK7 or FN1. PARK7 and FN1 band intensities were quantified by densitometric scanning and relative secretion level to vehicle-treated cells is shown. n = 3; **, p < 0.01; n.s., not significant. (D) SH-SY5Y cells were homogenized by using the Dounce homogenizer and homogenate was sequentially centrifuged as indicated. Equal aliquots from each fraction were immunoblotted using antibodies specific for PARK7, LMNA (lamin A/C), VDAC1, RPN1, or proCASP3 (caspase 3). (E) SH-SY5Y cells were cultured in serum-free medium for 3 h. Whole cell lysates and the conditioned medium were separated by 2D-PAGE and immunoblotted using antibody specific for PARK7. The ratio of oxPARK7 to total PARK7 is shown under each condition.
secretion was caused by another neurotoxin-induced oxidative stress. We tested the mitochondrial complex I inhibitor rotenone, which can cause reactive oxygen species (ROS) production and a decrease in GSH levels [37]. We found that treatment with rotenone at non-toxic concentrations for 24 h induced PARK7 secretion (Fig. S1D).
To evaluate whether the oxidized form of PARK7 was preferentially secreted from cells treated with 6-OHDA, 2D-PAGE analysis was carried out to determine the ratio of reduced: oxidized PARK7. Results showed that the ratio of oxPARK7 to total PARK7 in cells was increased by 6-OHDA treatment (Figs. 1E and 3D). Furthermore, reduced and oxidized forms of PARK7 were both observed in medium, and the ratio of oxPARK7 to total PARK7 in medium was similar to that in cells. To further assess the involvement of Cys106 in PARK7 secretion, HEK293 cells were transfected with either wild-type (WT) PARK7 or PARK7 C106S , the latter being a mutant form of PARK7 in which oxidation-sensitive cysteine at amino acid 106 is substituted by serine. Results of transfection indicated that 6-OHDA treatment induced PARK7 C106S secretion in a manner similar to that observed with endogenous PARK7 and WT PARK7 (Figure 3(E)). Together, the foregoing series of results suggest that oxidation of PARK7 at Cys106 is not involved in enhancement of PARK7 secretion.

6-OHDA induces autophagy in SH-SY5Y cells and MEF cells
To gain insight into the mechanism by which 6-OHDA treatment increased PARK7 secretion, we examined whether 6-OHDA induced autophagy, since it had recently been shown that the secretion of several proteins lacking a signal sequence is associated with the autophagy-based unconventional secretory pathway [38][39][40]. To exclude any possible effect of incubation of cells in serum-free medium on monitoring of autophagy induction, autophagic flux was evaluated using the lysate from the cells treated with 6-OHDA for 3 h. By monitoring the conversion of MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) from a cytosolic form (LC3B-I) to its lipidated phagophore-and autophagosome-associated form (LC3B-II), we found that 6-OHDA treatment induced increase in LC3B-II levels in SH-SY5Y cells (Figure 4(A)).
In this context, to determine whether autophagy could be induced under the same conditions for which we had observed that 6-OHDA treatment increased PARK7 secretion, we assessed the effects of 6-OHDA on PARK7 secretion and autophagy induction in WT MEF cells, which have been widely used in autophagy research. We found that (B) PARK7 band intensities were quantified by densitometric scanning and the percentage of secreted PARK7/total PARK7 is shown. LDH release in the conditioned medium was analyzed by LDH assay. n = 3; mean ± S.D.; *, p < 0.05. (C) SH-SY5Y cells were pre-treated with 2 μg/ml brefeldin A for 3 h and were then treated with 100 μM 6-OHDA for 3 h, followed by culture in serum-free medium for 2 h. Whole cell lysates and the conditioned medium were immunoblotted with antibodies specific for PARK7, FN1, or RPN1. PARK7 and FN1 band intensities were quantified by densitometric scanning and relative secretion level to vehicle-treated cells is shown. n = 3; mean ± S.D.; **, p < 0.01; n.s., not significant. (D) SH-SY5Y cells were pre-treated with or without 20 μM pan-caspase inhibitor (zVAD) or CASP1 inhibitor (YVAD) for 1 h and were then treated with 100 μM 6-OHDA for 3 h, followed by culture in serum-free medium for 2 h. Whole cell lysates and the conditioned medium were immunoblotted using antibodies specific for PARK7 or RPN1. PARK7 band intensities were quantified by densitometric scanning and relative secretion level to vehicle-treated cells is shown. n = 3; mean ± S.D.; **, p < 0.01.
6-OHDA treatment increased PARK7 secretion in MEF cells, although the amount of secreted PARK7 from 6-OHDA-treated MEF cells was less than that from 6-OHDA-treated SH-SY5Y cells ( Figure 4(B)). Significant LDH release was not observed in cells treated with 100 μM 6-OHDA (data not shown). We further found that significant increase in LC3B-II levels was observed in cells treated with 75 μM 6-OHDA ( Figure 4(C)). Since it had previously been indicated that an increase in LC3B-II level may be associated with either enhanced formation of autophagosomes or impairment of autophagosome turnover [41], we decided to evaluate the effect of co-treatment of 6-OHDA with bafilomycin A 1 , which inhibits acidification inside the lysosome and to block fusion between the autophagosome and the lysosome. Results showed that LC3B-II levels increased in 6-OHDA-treated cells, and that this increase was enhanced by co-treatment of 6-OHDA with bafilomycin A 1 , indicating that there was increase in autophagic flux during 6-OHDA treatment in MEF cells (Figure 4(D)). We also evaluated the effect of bafilomycin A 1 on autophagic degradation and on secretion of PARK7, finding as a result that although LC3B-II levels increased in response to treatment with bafilomycin A 1 alone ( Figure (4)), this did not result in induction of PARK7 secretion (Figure 4(E)). We further found that this increase in 6-OHDA-induced PARK7 secretion was suppressed by co-treatment with bafilomycin A 1 , suggesting the possibility that autophagic flux is important for PARK7 secretion and that PARK7 is not a substrate for autophagic degradation. We also used a fluorescent probe for lysosomes, LysoTracker Red, to evaluate the lysosomal membrane integrity. Decreased fluorescence of LysoTracker Red reflects lysosomal membrane permeabilization and/or an increase in lysosomal pH. As control, bafilomycin A 1 treatment caused a decrease in LysoTracker Red fluorescence both in SH-SY5Y cells (Fig. S2A) and WT MEF cells (Fig. S2B), as expected. In contrast, the staining pattern of LysoTracker Red puncta in 6-OHDA-treated cells was similar to that in vehicle-treated cells. The lysosomal membrane integrity was further evaluated by subcellular fractionation. Results showed that in vehicle-treated cells, lysosomal protease CTSB (cathepsin B) was found almost exclusively in the membrane organellar protein-rich NP-40 extract but not in the cytosolic protein-rich digitonin extract (Fig. S2C). 6-OHDA treatment did not significantly change the localization of CTSB. Together, these results suggest the conditions in which 6-OHDA induced secretion of PARK7 do not affect lysosomal membrane integrity. We also performed transmission electron microscopy analysis to elucidate the induction of autophagy in 6-OHDAtreated WT MEF cells. Results indicated that while there Together, these results suggest that the same conditions in which 6-OHDA induces secretion of PARK7 also induce autophagy.

PARK7 secretion is suppressed in autophagy-deficient MEF cells and ATG16L1 knockdown SH-SY5Y cells
To further examine the involvement of autophagy in 6-OHDA-induced PARK7 secretion, we investigated the response to 6-OHDA in Atg5-deficient (atg5 -/-) MEF cells, as ATG5 is known to play an essential role in autophagosome formation [42]. Results for atg5 -/-MEF cells indicated that there was no LC3B-II formation and that 6-OHDA did not induce PARK7 secretion ( Figure 5(A)). We further employed Atg9-deficient (atg9 -/-) and Atg16l1-deficient (atg16l1 -/-) MEF cells, in both of which autophagosome formation was  Whole cell lysates were immunoblotted using antibodies specific for LC3B or ACTB. LC3B and ACTB band intensities were quantified by densitometric scanning and LC3B-II:ACTB ratio is shown. n = 3; mean ± S.D.; **, p < 0.01. (B) WT MEF cells were treated with 0-100 μM 6-OHDA for 3 h and were then cultured in serum-free medium for 2 h. Whole cell lysates and the conditioned medium were immunoblotted using antibodies specific for PARK7 or ACTB. PARK7 band intensities were quantified by densitometric scanning and the percentage of secreted PARK7/total PARK7 is shown. LDH release in the conditioned medium was analyzed by LDH assay. n = 3; mean ± S.D.; *, p < 0.05. (C) WT MEF cells were treated with 75 μM 6-OHDA for 3 h. Whole cell lysates were immunoblotted using antibodies specific for LC3B or ACTB. LC3B band intensities were quantified by densitometric scanning and LC3B-II:LC3B-I or LC3B-II:ACTB ratio is shown. n = 3; **, p < 0.01. (D and E) WT MEF cells were pretreated with 100 nM bafilomycin A 1 (BafA1) for 1 h and were then treated with 75 μM 6-OHDA for 3 h (D), followed by culture in serum-free medium for 2 h (E). Whole cell lysates and the conditioned medium were immunoblotted using antibodies specific for LC3, PARK7, or ACTB. PARK7, LC3B and ACTB band intensities were quantified by densitometric scanning. LC3B-II:ACTB ratio (D) and relative secretion level to vehicle-treated cells (E) are shown. n = 3; mean ± S.D.; **, p < 0.01. (F) WT MEF cells were treated with vehicle (i) or 75 μM 6-OHDA (ii-v) for 3 h. Cells were subjected to electron microscopy. Representative images are shown. Black arrowheads indicate lysosome. Black arrows indicate autophagosome/amphisome and lysosome. White arrowhead indicates autolysosome. White arrow indicates phagophore. (G) WT MEF cells grown on coverslips were treated with 75 μM 6-OHDA for 3 h. Cells were processed for immunofluorescence staining with antibody against PARK7 or LC3B. Representative confocal images are shown. White arrows indicate co-localization. Scale bar: 10 μm.
To evaluate whether extracellular PARK7 was associated with small vesicles, we performed ultracentrifugation fractionation of conditioned medium at 100,000 x g to separate vesicle-associated proteins from soluble proteins. CAV1 (caveolin 1), secretion of which has been reported to occur with vesicles [45], was recovered from the pellet fraction, while PARK7 was recovered from the supernatant fraction, in both the medium collected from vehicle and 6-OHDA-treated cells (Fig. S3). These results suggest that extracellular PARK7 exists as soluble protein.

Oxidative stress is implicated in 6-OHDA-induced autophagy and PARK7 secretion
We then sought to test whether 6-OHDA-induced oxidative stress stimulated autophagy and PARK7 secretion. As had been observed in SH-SY5Y cells, we observed that GSH content was significantly reduced by 6-OHDA treatment in WT MEF cells (Figure 6(A)). We also used the fluorescent probe DCFH-DA to monitor intracellular ROS levels, finding as a result that treatment with 6-OHDA caused an increase in ROS levels as compared with vehicle-treated cells, and that this increase could be mitigated by treatment with NAC ( Figure 6(B)). We further found that 6-OHDA-induced PARK7 secretion could be suppressed by treatment with NAC ( Figure 6(C)) in a similar manner as had been observed in SH-SY5Y cells (Figure 3 (B)). Under conditions which were the same as the foregoing, we further noted that 6-OHDA induced an increase in LC3B-II levels, and that this increase could be suppressed by co-treatment with NAC ( Figure 6(D)). Together, these results suggest that 6-OHDA-induced oxidative stress is implicated in autophagy-based unconventional secretion of PARK7.
Activation of AMPK and ULK1 is implicated in 6-OHDAinduced autophagy and PARK7 secretion Because a cytotoxic concentration of 6-OHDA has been reported to induce AMPK-dependent autophagy in SH-SY5Y cells [46], we examined whether activation of the AMPK pathway could be made to occur under the same conditions as those for which we had observed that 6-OHDA caused secretion of PARK7. We investigated the effect of 6-OHDA on phosphorylation of AMPK at Thr172 (AMPK activation) and on phosphorylation by AMPK of its downstream effector ULK1 at Ser555 (ULK1 activation). Since the inhibitory function of MTOR in autophagy is well-established [41], the phosphorylation of RPS6KB1/p70S6K (ribosomal protein S6 kinase B1) at Thr389 by MTOR was also evaluated to assess MTOR activity. As a control, the MTOR inhibitor rapamycin was employed, this being observed to cause a decrease in RPS6KB1 phosphorylation without causing stimulation of AMPK and ULK1 phosphorylation (Figure 7(A)), as expected. In contrast, 6-OHDA treatment enhanced phosphorylation of AMPK and its substrate ULK1, but did not significantly change the degree of phosphorylation of RPS6KB1. We therefore evaluated the effect of NAC on AMPK phosphorylation, finding as a result that NAC treatment significantly diminished AMPK phosphorylation in a manner roughly paralleling the observed decrease in LC3B-II levels (Figure 7(B)). Because AMPK activation is regulated by the AMP:ATP ratio, we assessed AMP and ATP levels, upon which we found that 6-OHDA treatment increased the AMP:ATP ratio (Figure 7(C)). These results suggest that the same conditions which exist when 6-OHDA induces oxidative stress-mediated PARK7 secretion and autophagy also cause activation of the AMPK-ULK1 pathway without affecting MTOR activity.
To confirm the involvement of the AMPK-ULK1 pathway in 6-OHDA-induced autophagy and PARK7 secretion, SH-SY5Y cells were treated with MRT68921, a specific inhibitor of ULK1 [47], as a result of which it was found that 6-OHDA-induced increase in LC3B-II levels was suppressed by co-treatment with MRT68921 (Figure 7(D)). Furthermore, 6-OHDA-induced PARK7 secretion was suppressed by co-treatment with MRT68921 (Figure 7(E)). Consistent with the results obtained for SH-SY5Y cells, 6-OHDA-induced PARK7 secretion was suppressed in WT MEF cells treated with MRT68921 (Figure 7(F)). Together, these results suggest that AMPK-ULK1 pathway is involved in 6-OHDA-induced autophagy and PARK7 secretion.

Trehalose but not rapamycin promotes PARK7 secretion in WT MEF cells
We then investigated whether another stimulator of autophagy could induce PARK7 secretion. In WT MEF cells, rapamycin treatment increased LC3B-II levels both in the presence and absence of bafilomycin A 1 as expected (Figure 8(A)). When the cells were treated with 1-5 μM rapamycin, an increase in PARK7 secretion was observed, but not to the same degree as had been observed with 6-OHDA treatment (Figure 8(B)). We next evaluated the effect of trehalose. Trehalose is a natural disaccharide found in many non-mammalian species that induces MTORindependent autophagy by inhibiting hexose uptake and subsequently activating the AMPK-ULK1 pathway [48,49]. It should be noted that it has recently been reported that release of SNCA (synuclein alpha) is upregulated by trehalose treatment in rat , followed by culture in serum-free medium for 2 h (E). Whole cell lysates and the conditioned medium were immunoblotted using antibodies specific for LC3, PARK7, or ACTB. LC3B, PARK7 and ACTB band intensities were quantified by densitometric scanning. LC3B-II:ACTB ratio (d) and relative secretion level to vehicle-treated cells (e) are shown. n = 3; mean ± S.D.; **, p < 0.01. (F) WT MEF cells were pre-treated with or without 2 μM MRT68921 for 30 min and were then treated with 75 μM 6-OHDA for 3 h, followed by culture in serum-free medium for 2 h. Whole cell lysates and the conditioned medium were immunoblotted using antibodies specific for PARK7 or ACTB. PARK7 band intensities were quantified by densitometric scanning and relative secretion level to vehicle-treated cells is shown. n = 3; mean ± S.D.; **, p < 0.01. pheochromocytoma PC12 cells [50]. When WT MEF cells were treated with trehalose, we found that LC3B-II levels were increased in a concentration-dependent manner (Figure 8(C)), confirming that there was activation of autophagic flux under the conditions tested. Furthermore, 200 mM trehalose enhanced PARK7 secretion at the same level with 6-OHDA treatment (Figure 8(D)) without significant LDH release (data not shown). Together, these results suggest the possibility that 6-OHDAinduced PARK7 secretion is mainly mediated by MTOR-independent autophagy.

Discussion
In our present study, we demonstrate that PARK7 is secreted via the autophagy-mediated unconventional secretion mechanism. Autophagy is generally understood to be the process by which cytosolic proteins and damaged organelles are degraded within lysosomes [41]. During macroautophagy, which is the most prevalent form of autophagy, some cytoplasmic components including organelles are enclosed within a double-membraned structure referred to as an autophagosome. The autophagosome undergoes the fusion with a lysosome (autolysosome) to achieve degradation of its internal contents. The autophagosome can also fuse with an endosome to form an amphisome before fusion with a lysosome. To understand the unconventional secretion mechanism, autophagy may be the mechanism by which cytosolic proteins are secreted into the extracellular space across the hydrophobic environment of the plasma membrane. It is conceivable that some form of autophagic structure (autophagosome, amphisome, or autolysosome) may fuse with the plasma membrane to release the enclosed cytosolic contents into the extracellular space. Indeed, autophagy-mediated unconventional secretion, termed secretory autophagy, has been implicated in the secretion of a subset of unconventionally secreted proteins which include yeast Acb1/DBI, mammalian IL1B, and IDE (insulin degrading enzyme) [8,[38][39][40]. Our study indicates that PARK7 is also secreted via the secretory autophagy. It is however not clear how such a secretory protein might be selected for secretory autophagy and escape lysosomal degradation. One possibility is that the secretion rather than degradation of cytosolic constituents that occurs in secretory autophagy may be induced by a shared but partially divergent autophagic machinery. In our present study, we demonstrated that 6-OHDA-induced PARK7 secretion required the autophagy-related proteins ATG5, ATG9, and ATG16L1, suggesting that ATG proteins which govern biogenesis of autophagic membranes are needed for secretory autophagy-mediated PARK7 secretion. Interestingly, we also showed that 6-OHDA induced MTOR-independent autophagy via the AMPK-ULK1 pathway. We furthermore observed that the MTOR-independent autophagy inducer trehalose caused PARK7 secretion to a degree on the same order as that observed with 6-OHDA treatment, but that the MTOR inhibitor rapamycin only poorly stimulated PARK7 secretion as compared with 6-OHDA treatment.
Since MTOR senses cellular nutritional status and is involved in the induction of degradative autophagy that occurs in response to nutrient starvation [41], understanding the role that MTOR plays should assist in elucidating the respective contributions of the MTOR signaling pathway and the MTOR-independent signaling pathway to secretory autophagy. Several cellular stresses, e.g., inflammation, nutrient stress, and ER stress, have been reported to trigger secretory autophagy [8,[38][39][40]. In the present study, we demonstrated that oxidative stress induced by 6-OHDA also triggered secretory autophagy. Oxidative stress has been implicated in the induction of degradative autophagy [51]. It has been reported that oxidation of Atg4 at cysteine inactivates its delipidating activity on LC3, thereby leading to the induction of autophagosome formation [52]. It has also been proposed that AMPK may be activated in response to increased intracellular ROS levels, particularly through S-glutathionylation [53]. 6-OHDA has been reported to impair the mitochondrial respiratory chain, leading to a decrease in ATP levels [33]. We therefore concluded that 6-OHDA-induced oxidative stress, and the resulting increase in the AMP:ATP ratio and AMPK-ULK1 activation, were implicated in PARK7 secretion at least under the conditions tested. Indeed, we demonstrated in the present study that ULK1 inhibition suppressed 6-OHDA-induced PARK7 secretion. Because the AMPK inhibitor compound C has been reported to induce autophagy in some type of cells [54], it will be necessary to pay attention to evaluate the effect of AMPK inhibition by compound C or AMPK siRNA on PARK7 secretion.
Our study suggests that AMPK-ULK1-mediated but MTOR-independent signaling regulation plays an important role in the distinctive induction of secretory autophagymediated PARK7 secretion. In addition, we speculate that a dedicated molecular machinery for formation or intracellular trafficking of autophagic structures may account for the distinctive secretory pathway of PARK7. Further investigations to identify the involvement of other autophagy-related proteins in PARK7 secretion are required for in-depth analysis. It should be noted that Galluzi et al. encourage the use of molecularly-oriented descriptors for non-degradative functions of autophagic machinery [55]. In line with this definition, the mechanisms of PARK7 secretion may need to be referred to as ATG5, ATG9, and ATG16L1-dependent secretion instead of secretory autophagy-mediated secretion. In future studies, it would also be of interest to evaluate whether other known key proteins involved in secretory autophagy, such as RAB8A (RAB8A, member RAS oncogene family) and GORASP2/GRASP55 (golgi reassembly stacking protein 2) [39,40], are implicated in PARK7 secretion, and whether 6-OHDA-induced secretory autophagy is implicated in the secretion of other proteins. It has been recently reported that dedicated SNAREs participate in secretory autophagy-mediated unconventional secretion of IL1B in response to lysosome damage [56]. Interestingly, secretion of IL1B depends on plasma membrane STX3 (syntaxin 3) and STX4 (syntaxin 4) instead of delivery to lysosomes via STX17 (syntaxin 17), which has been identified as a necessary protein for autophagosomal fusion with the endosome/lysosome [57]. To consider how autophagy-mediated PARK7 secretion separates from degradative autophagy, further investigations to elucidate the possible involvement of dedicated SNARE machinery in PARK7 secretion may provide another insight.
With respect to the differences between target proteins for degradative versus secretory autophagy, it is possible that secretory proteins in autophagic vacuoles may possess some protective modification or domain that contributes in some way to prevent lysosomal degradation. It has been demonstrated that the SlyX domain (EKPPHY) of IDE contributes to IDE secretion by preventing its lysosomal degradation [40,58]. Furthermore, KFERQ-like motifs of IL1B are thought to be required for entry of IL1B into the lumen of the phagophore so as to permit secretion as a soluble protein either through a direct fusion of the autophagosome with the plasma membrane or via the multi-vesicular body pathway [59]. It is also known that a KFERQ-like motif is present in substrates of chaperonemediated autophagy [41]. In the case of PARK7, we found that there are several KFERQ-like motifs in the PARK7 amino acid sequence. Because we found in the present study that PARK7 secretion is mediated by secretory autophagy, studies to elucidate the possible involvement of KFERQ-like motifs in PARK7 secretion may provide further insight.
Electron microscopy analysis showed that 6-OHDA treatment induced formation of structures typically observed where there is involvement of the autophagic pathway, such as autophagosomes, amphisomes, and autolysosomes. Since ATG5, ATG9, and ATG16L1 are known to play essential roles in autophagosome formation [41][42][43][44], one possibility is that cytosolic PARK7 may be packaged within LC3-positive autophagic vacuoles for secretion. Indeed, in addition to cytosolic distribution of PARK7 signal, we observed the modest but significant localization of PARK7 in LC3B-positive puncta in the cells treated with 6-OHDA. Since our rough estimate of PARK7 band intensity suggested that less than 10% of PARK7 was secreted from cells in response to 6-OHDA treatment, we posit that this might affect the modest distribution of PARK7 in autophagic vacuoles. Since ultracentrifugation fractionation of conditioned medium showed that extracellular PARK7 was not associated with exosomes or other such small vesicles, we speculate that the PARK7containing autophagic vacuole, if it exists, is most likely fusing with the plasma membrane to cause release of its soluble contents including PARK7 into the extracellular space, but we cannot exclude the possibility that PARK7 is first secreted via exosomes/ectosomes and then released from vesicle in the extracellular space.
Several lines of evidence suggest that secreted PARK7 may play a functional role in the extracellular space. For example, PARK7 secreted from osteoblasts has been found to promote angiogenesis and osteogenesis [32]. Extracellular deposition of abnormal TTR (transthyretin) protein has been shown to cause familial amyloid polyneuropathy, and secreted PARK7 has been shown to degrade aggregated TTR to protect against the onset of amyloid polyneuropathy [26]. And with respect to PD, PARK7 has been detected in the cerebrospinal fluid of patients suffering from PD [28,29], and it has been found that administration of recombinant PARK7 into the brain of 6-OHDA-induced PD rat model improved PD phenotype [60], suggesting that extracellular PARK7 might exert a protective role against PD. There is a possibility that secreted PARK7 may protect neighboring cells from oxidative stress by means of a process similar to paracrine signaling, which possibility is thought to be supported by reports that PARK7 knockout astrocytes show inferior ability to protect neuronal cells against 6-OHDA-induced cell death both by co-culture and through astrocytes conditioned medium [61]. Since PARK7 has 3 reactive cysteine residues, it is also possible that secreted PARK7 itself receives oxidative modification by scavenging ROS within the extracellular space. Indeed, when the conditioned medium obtained from non-treated cells was incubated in the presence of 6-OHDA, the ratio of oxPARK7 to total PARK7 was increased, suggesting that oxidation of PARK7 occurred in the presence of 6-OHDA within the extracellular space (Fig. S4).
Here we have presented results indicating that PARK7 is secreted via the autophagy-based unconventional secretory pathway. In summary, our data suggest that electrophilic 6-OHDA quinone lowers GSH levels and induces oxidative stress, and that this is followed by AMPK-ULK1 activation to induce secretory autophagy, resulting in unconventional secretion of PARK7 (Figure 9).  . The protein samples were subjected to SDS-PAGE or 2D-PAGE, and to immunoblotting using appropriate antibodies as indicated in figures. An LDH (lactate dehydrogenase) release assay was performed as described previously [63]. Data are expressed as percentage of total LDH activity, after subtraction of background as determined from the medium alone.

2D-Page
Trichloroacetic acid-precipitated whole cell lysates or medium were mixed with rehydration buffer (

Subcellular fractionation
SH-SY5Y cells were homogenized using a Dounce homogenizer with 60 strokes in 200 μl homogenization buffer (250 mM sucrose, 20 mM Tris-HCl, pH 7.4, 1 mM EDTA-2Na) with protease inhibitor cocktail as described previously [64]. The resulting homogenate was centrifuged at 800 x g for 10 min twice at 4°C. The post-nuclear supernatant was pelleted at 8,000 x g for 10 min twice at 4°C. The resulting supernatant was further centrifuged at 100,000 x g for 30 min in a Beckman TLA100.3 rotor at 4°C. Each pellet was resuspended in 200 μl homogenization buffer. Equal aliquots from each fraction were analyzed for immunoblotting. Crude cytosolic and membrane/organelle fractions were isolated from WT MEF cells by sequential detergent extraction as described previously [65]. Briefly, cells were resuspended by gentle pipetting in 200 μl digitonin buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 40 μg/ml digitonin [Wako, 043-21371]). After 10 min incubation on ice, the cell suspension was centrifuged at 8,000 x g for 5 min at 4°C. The resulting supernatant was kept as cytosol-enriched digitonin extract. The resulting pellet was resuspended by vortexing in 200 μl NP-40 buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1% NP-40). After 30 min incubation on ice, samples were centrifuged at 13,000 x g for 5 min at 4°C. The resulting supernatant was kept as membrane organellar protein-enriched NP-40 extract. Equal aliquots from each fraction were analyzed for immunoblotting.

Detection of lysosome or intracellular ROS by fluorescence microscopy
For lysosome staining, cells grown on coverslips were treated with 6-OHDA or bafilomycin A 1 for 3 h. Cells were then stained with LysoTracker Red DND-99 (Thermo Fisher Scientific, L7528) at a final concentration of 500 nM in serum-free medium for 1 h at 37°C. To detect intracellular ROS generation, cells were pre-incubated with 10 μM of DCFH-DA (Thermo Fisher Scientific, D399) for 1 h before 6-OHDA treatment. Cells were fixed with 4% paraformaldehyde (Wako, 166-2325)/PBS for 20 min and were washed with PBS twice. Cell nuclei were stained with Hoechst 33342 (Dojindo, 346-07951). Fluorescence was detected using a fluorescence microscope (OLYMPUS IX71).

Immunofluorescence staining
Cells grown on glass coverslips were fixed with cold methanol for 10 min at 4°C, and then blocked with 3% BSA in PBS for 1 h. The coverslips were incubated with anti-PARK7 (3843) and anti-LC3B antibodies for 3 h at room temperature. Confocal fluorescence images were obtained using a Zeiss LSM710 confocal microscope with oil objective lens and accompanying software (LSM Software ZEN2009).

Electron microscopy analysis
For electron microscopy, cells were fixed in phosphate-buffered 2% glutaraldehyde (Electron Microscopy Sciences, 16220), and subsequently post-fixed in 2% osmium tetroxide (Nisshin EM, 300-1) for 3 h in an ice bath, following which the cells were dehydrated in ethanol and embedded in epoxy resin. Ultrathin sections were obtained using the ultramicrotome technique. Ultrathin sections stained with uranyl acetate (Cerac) for 10 min and with lead staining solution (Sigma-Aldrich, 18-0875-2) for 5 min were subjected to TEM observation (JEM-1200 EX, JEOL) at Hanaichi Ultrastructure Research Institute (Okazaki, Japan).

Knockdown of ATG16L1 by small interfering RNA
ON-TARGETplus human ATG16L1 siRNA (L-021033) or Non-targeting siRNA (D-001810) were obtained from Dharmacon. SH-SY5Y cells grown in 6-well plates were transfected with siRNA, at a concentration of 30 pmol/well with Lipofectamine RNAiMAX (Thermo Fisher Scientific, 13778) twice according to the manufacturer's instructions. Seventytwo h after transfection, cells were used for further analyses.

Determination of cellular AMP:ATP ratio
Cells were lysed in 0.25 ml of 0.6 N perchloric acid. Acidinsoluble material was removed by centrifugation at 1,000 x g for 5 min. The supernatant was adjusted with KOH to pH 5.0-7.0 and, after 10 min, was centrifuged at 8,000 x g for 5 min to remove KClO 4 . HPLC analysis was carried out using an Inertsil ODS-2 column (GL Science). Flow rate of eluent (0.1 M sodium phosphate buffer, pH 6) was 0.5 ml/min. Nucleotides were detected by their absorbances at 260 nm, AMP:ATP ratios being calculated using standards for AMP and ATP.

Statistical analysis
Data are reported as mean ± S.D. of at least 3 independent experiments unless otherwise indicated. Statistical processing was conducted by variance analysis using Tukey test for multiple comparisons and Student t-test for comparison of 2 means. The difference was considered significant when the p value was < 0.05.


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Identification and DNA annotation of a plasmid isolated from Chromobacterium violaceum

Chromobacterium violaceum is a ß-proteobacterium found widely worldwide with important biotechnological properties and is associated to lethal sepsis in immune-depressed individuals. In this work, we report the discover, complete sequence and annotation of a plasmid detected in C. violaceum that has been unnoticed until now. We used DNA single-molecule analysis to confirm that the episome found was a circular molecule and then proceeded with NGS sequencing. After DNA annotation, we found that this extra-chromosomal DNA is probably a defective bacteriophage of approximately 44 kilobases, with 39 ORFs comprising, mostly hypothetical proteins. We also found DNA sequences that ensure proper plasmid replication and partitioning as well as a toxin addiction system. This report sheds light on the biology of this important species, helping us to understand the mechanisms by which C. violaceum endures to several harsh conditions. This discovery could also be a first step in the development of a DNA manipulation tool in this bacterium.

SCIENTIFIC RepoRts | (2018) 8:5327 | DOI: 10.1038/s41598-018-23708-5 re-suspended with 1 mL of TE Buffer and one volume of phenol:chloroform was added. After centrifugation (22789 × g, 10 minutes, 4 °C), the aqueous phase was transferred to a new tube and the DNA was precipitated with one volume of isopropanol. Finally, the DNA was washed with 80% Ethanol and the four independent preparations were re-suspended with 500 μL of TE.
In order to certify that our preparation was free of genomic DNA we isolated the band containing the plasmid and digested the agarose using ß-agarase (NEB catalog # -M0392S) according to the manufacturer's instructions.
Optical analysis of DNA in nanochannels. The optical DNA mapping of the single plasmid molecules were performed as described in ref. 18 . Using a combination of a DNA fluorescent dye (YOYO-1) and Netropsin, an antibiotic that binds specifically to AT DNA regions, this technique allows the acquisition of DNA barcode images, with dark and bright regions corresponding to AT-rich and GC-rich regions respectively 19 . In this way, the pattern of the emission intensity reflects the sequence of the DNA molecule, with a resolution on the kilobasepair length scale. The nanofluidic chips were fabricated in fused silica, using conventional techniques, as described in ref. 20 . All the data was recorded, using a Zeiss AxioObserver. Z1 microscope equipped with a 100× TIRF oil immersion objective (NA = 1.46) from Zeiss and a Photometrics Evolve EMCCD camera.
NGS Sequencing and Assembly. The DNA was quantified using Qubit Fluorometric Quantitation and the quality was checked on an agarose gel. The library was prepared using TruSeq Nano DNA Sample Preparation Kit (Illumina) according to the manufacturer's instructions and then sequenced on the Illumina MiSeq at Fasteris SA. For the base-calling, the CASAVA pipeline 1.8 was used. De novo genomic assembly was made using VELVET v1.2.10 and Burrows-Wheeler Alignment Tool (v0.5.9) for mapping.
Plasmid annotation and comparison. The annotation was made using Glimmer (v3.02b), a software built to find genes in bacteria, archaea and viruses. Bacteria/archaea genetic code and circular topology were chosen. The search for homology of the whole pChV1 sequence was made using the BLASTn program against non-redundant (NR) NCBI database and against a specific bacteriophage database (unclassified bacteriophages -taxid: 12333), also from NCBI. Comparison of the predicted ORFs in genomic databases was made using BLASTx. Hits with more than 50% coverage and with the highest BitScore were picked. Search for tRNAs was made using the online version of tRNAscan-SE v1.21 in default mode. DNA inverted repeated sequences were obtained using Einverted (http://emboss.bioinformatics.nl/cgi-bin/emboss/einverted). The search for palindromic DNA was made using the MEME web-tool 21 . GC content profile and GC-skew were obtained using GC-Profile 22 and GenSkew (http://genskew.csb.univie.ac.at/), respectively. Data availability. The pChV1 complete sequence is available at GenBank (accession number -MG651603).
FASTQ file is also available in the Sequence Read Archive (SRA) repository with accession number SRR6363036.

Results
Identification of an episome in C. Violaceum strain ATCC 1242. While extracting genomic DNA from C. violaceum strain ATCC 12472 to construct a genomic library, we noticed after agarose gel electrophoresis the recurrence of a DNA species smaller than expected for high molecular weight genomic DNA in our preparations. We hypothesized that this DNA species could be a circular episome. We therefore carried out standard plasmid DNA preparations and analyzed the purified DNA by agarose gel electrophoresis and ethidium bromide staining. As can be seen in lane 2 of Fig. 1, the preparation contained contaminating high molecular weight genomic DNA trapped in the well but also a species with mobility much greater than 10 kb, our putative episome  Competitive binding was used in order to produce the emission intensity pattern along the linear for of the plasmid. as indicated by a star symbol. A third faster migrating species was also observed. We next performed a restriction enzyme analysis of our preparation with KpnI, BamHI or EcoRI ( Fig. 1 lanes 3 -5). Consistent with linearization of a circular DNA molecule, digestion with KpnI resulted in a single band and disappearance of genomic DNA, both the DNA trapped in the well and the third species described above, due to characteristic smearing of genomic DNA digestions ( Fig. 1 lane 3). Digestion with BamHI or EcoRI resulted instead in defined patterns of discrete DNA fragments ( Fig. 1 lanes 4 and 5). We then performed the same analysis using seven different additional C. violaceum strains (Fig. 2). Preparations from strains CVAC02, CVAC05, and CVT8 appeared to contain a putative episome similar to strain ATCC 12472, while preparations from strains CV026, CVT19 and CVRP5 appeared to contain only genomic DNA. The preparation from strain CVT24 also seemed to contain a putative episome species but the result from the restriction analysis is difficult to interpret. Thus, we identified an episome in C. violaceum strain ATCC 12472 and propose to name it pChV1.
Episome pChV1 is a circular plasmid. The above restriction enzyme analysis suggested that pChV1 is a circular DNA molecule. To verify this hypothesis the episome DNA was purified after gel electrophoresis ( Fig. 1 lane 2, band indicated with a star symbol) and stained with YOYO-1 (a bis-intercalator fluorescent dye) and Netropsin (a minor groove binder of AT-rich sequences of double-stranded DNA) that competes with YOYO-1 intercalation 19 . Such stained preparations were diluted and injected in nanochannels to observe individual extended episome DNA molecules by fluorescence microscopy (Fig. 3A). Initially, the contour length of  The pChV1 DNA sequence. The complete sequence of pChV1 revealed a circular element with 44,212 bp with a G + C content of 65.96% (Table 1). 39 Open Reading Frames (ORFs) were found, which comprises 89,66% of the whole plasmid (Fig. 4). From these, 28 are conserved hypothetical proteins and 1 is a hypothetical protein.
Comparing the ORFs of the plasmid with other organisms, we observed that 17 (43%) of the ORFs have similarity with ORFs from Pseudogulbenkiania ferrooxidans. No tRNAs genes were found. We also searched for homology with bacteriophages and the BLAST analysis did not give any similarity with any phage genomes.  Plasmid maintenance genes. The plasmid has at least 4 known genes related to plasmid segregation/replication: parA, parB, repA and a gene with RPA domain, involved in plasmid replication initiation ( Table 2). parA and parB encode the ParA and ParB proteins, respectively. These proteins are part of the Type I plasmid-partitioning system and are responsible for ensuring the correct propagation of plasmids to daughter cells throughout cell division 24 . This partitioning system is founded in prophages, plasmids and chromosomes 25 .
RepA is a protein related to plasmid replication and is characteristic of P1 plasmids.
Structural phage genes. An abundant number of genes related to phage structure are present in the sequence of the plasmid. Genes that codify the baseplate, sheath and tail proteins as well as conserved hypothetical genes with domains related to phage structure are in close proximity in the pChV1 sequence.
Other genes. A DNA invertase (ORF_35), an enzyme that catalyzes site-specific recombination in phages was found. A conserved hypothetical protein (ORF_17) with a transposase domain is also present in the plasmid sequence. Toxins (ORF_13 and ORF_37) that may be related to the toxin-antitoxin (TA) system responsible for assuring the survival only for the cells with a copy of the lisogenyzed phage were also located. Other worthy-mention genes are: conserved hypothetical proteins with Ribonuclease E domain, XRE domain and ATPase AAA domain.
GC profile and GC-skew. We were able to identify two points in the sequence of pChV1 where the GC content drops when comparing to the whole sequence (Fig. 5). These variations also qualitatively agree with intensity variaitons in the single molecules studies in Fig. 3. This might reflect the presence of two origins of replication that are present in P1-like plasmids, oriR and oriL. GC-skew also helps predicting the location of the leading and lagging strand and cumulative GC-skew values reflect the origin and terminus points of replication 26 . In our analysis, we can observe throughout the cumulative GC-skew curve, two regions that we could call minimum points that sign the origins of replication oriR and oriL (Fig. 6).
Repeated and palindromic sequences. A 19 bp inverted repeated sequence separated by 1,785 bp was also located and may be involved in the circularization of the phage or other homologous recombination-based process (Table 3). This pair of sequences is located flanking the partitioning related genes parA and parB (Fig. 7).
Other inverted repeat sequences with size varying from 23 to 54 bp were also founded although the complementarity between the pair of repeats was not 100% (data not shown). Palindromic sequences located at two distinct sites in the sequence and varying from 7 to 17 bp are also present (Table 3).

Discussion
The first bacteriophage was discovered in the 1950's 27 and since then, the number and variety of new viruses that infect bacteria has grown considerably, reaching more than 1,300 genome projects according to the NCBI database. While researching the opportunistic pathogen C. violaceum, in genomic preparations we observed an extra-chromosomal DNA of high molecular weight (but lower than it would be if it was genomic DNA). We then isolated and sequenced this putative plasmid which proved to have genes from the P1 bacteriophage/plasmid group.   Table 3. Pairs of Inverted repeated and palindromic sequences founded in pChV1. After the sequencing of C. violaceum 7 , the presence of four different sequences of prophages (CvP1-4) were observed in the C. violaceum's genome 28 . Neither of these is related to the plasmid we report here. Before this, tail-like particles were observed in C. violaceum by electron microscopy although no biological activity was associated to them 29,30 .
According to the sequence data and annotation, the plasmid founded in C. violaceum could be a P1-like virus due to the presence of genes that encode for structural viral particles. Moreover, genes related to plasmid partitioning and the plasmid initiator protein RepA are strong evidence to classify this plasmid as a P1-like phage. Another hallmark of P1-like phages is the presence of toxin-antitoxin genes that constitute a plasmid addiction system. In pChV1 two ORFs are predicted to be toxin genes (ORFs 13 and 37 with 52% and 90% of identity, respectively) although further studies need to be done to confirm the presence of this system. From our search for homology, we observed that pChV1 has a nucleotide sequence very different from other phages described so far. This feature hampers the search for phage-related sequences, such as lox sites, incC and incA and others, which are, in general, well conserved between other viruses, but does not exclude the existence of them in pChV1. However, repeated sequences that are founded amongst other phages are also present in pChV1, such as the 19 bp inverted repeated sequence (Fig. 7).
Origins of replication are GC-poor regions and locating them in the plasmid may suggest the locals where replication starts. Although we were not able to predict specific sequences that would correspond to origins of replications in pChV1, the GC content profile and GC-skew showed two regions that might reflect oriR and oriL. oriR, that is used during plasmid maintenance replication, is in the same region as the parA, parB and repA genes. This co-location of a possible origin of replication and the plasmid maintenance genes is observable in pChV1. Conversely, oriL is related to lytic growth and is separated about 9 kb from oriR in P1 31 . We suggest that the second possible origin of replication founded in pChV1 (located approximately at 27 kbp) corresponds to oriL.
One notable feature is that when we aligned the predicted open reading frames using BLASTn (that searches a nucleotide query in a nucleotide database) we obtained no significant result. Conversely, when BLASTx was used (searches a translated nucleotide query in a protein database) we were able to identify genes with high degree of confidence. This means that during evolution this virus accumulated many mutations on its DNA sequence but conserved -to some extent -the amino acid composition of its proteins. For example, pChV1 has many ORFs with more than 90% of identity with other genes found in bacteria ( Table 2). When we aligned these same ORFs using BLASTn we did not obtain any significant result.
Besides the presence of phage-related genes and sequences, some essential elements that would make pChV1 a functional P1-phage are still missing 31 . By the lack of evidence, we cannot conclude if this plasmid is a temperate P1-like phage, or if it is a chimeric DNA, part bacteriophage or plasmid. Moreover, it could be a fragment of DNA that is maintained inside C. violaceum by addiction systems but defective in its capacity of lisogeny. Conversely, the tail-like particles observed in the 1970s 29,30 could be an evidence that, under stress, the phage proteins encoded by pChV1 would be produced.
Genetic mobile elements are still important in the field of molecular biology. Beside this, the use of phage-derived systems as tools has allowed genome manipulation of all kind of organisms. In this way, further study of pChV1 would bring new ways to investigate genetic aspects of C. violaceum and maybe other species. Finally, pChV1 with its great number of hypothetical ORFs, is a rich reservoir of unexplored genes that might contribute to our understanding of the mechanisms underlying viral infections and plasmids.

Conclusion
In our work, we discovered an extra-chromosomal DNA -that we named pChV1 -in the opportunistic pathogen Chromobacterium violaceum. This plasmid is present as a low-copy plasmid and has most of its genetic apparatus composed of ORFs with unknown function, making pChV1 an important source of genes to be further explored. More than this, when its biology is better understood, this element can be used in genetic studies in C. violaceum as well as in other organisms.


Biochemical Signals and Material Characteristics

The chemical relationship between individual organisms and inside organisms is a universal law. Studies have found that plants can transmit disease-resistant information through volatile compounds. When plants are invaded by viruses or insects, they produce a series of secondary metabolites to fight against viruses or insects. At the same time, plants also produce volatile chemicals that spread through the air to nearby plants and induce them to produce system resistance; on the other hand, plants can also detect the type of invading insects and release specific odor compounds to attract insect predators. The study of volatile chemical substances transmitted between plants opens up new avenues for the development of new ecological agents and the prevention and control of plant diseases and insect pests.

Introduction
In the relationship between plants and animals, chemical links are also widespread. It is also a biochemical signal research area. The original form of the plant's defense system is generally the use of physical methods such as hair and thorns, followed by the synthesis of alkaloids and other compounds. The primary level of chemical defense is to make plants unpalatable, bitter, or difficult to digest; plant hormones (such as juvenile hormones) that can synthesize insects, and the growth and development of insects will be affected after feeding; the plants attract insects to spawn during flowering, At other times, the insects were rejected because of the difference in the proportion of the hexanol, the attractant, and the hexanal, the repellent, synthesized by plants at different times. Many relationships between plants and herbivores (especially insects) are chemically co-evolutionary. The feeding of insects promotes the development and differentiation of plant defense chemicals. The toxic components of plants also promote the development and detoxification of insects. mechanism. After being attacked by insects or infected by pathogens, plants can increase the concentration of existing defense substances or synthesize new chemicals. This phenomenon is called induced resistance. Induced resistance is a more effective and saved form of defense than conventional resistance. After the plant is harmed, it can release special volatile gases to attract natural enemies of pests. The latest study found that after tobacco is infected with tobacco mosaic virus, it produces a volatile compound called methyl salicylate, which is able to induce resistance in the neighboring tobacco.

Research on Human Biochemical Signal Substances
Acetone is produced in animals by two routes: deacetylation of acetoacetate and dehydrogenation of isopropanol. The former is the main way to produce acetone and occurs through lipolysis or amino acid degradation. The mice were fed 2-14C-acetone by gastric tube or injection. The results showed that acetone could be metabolized by acetate and formate. For licking milk animals, when the metabolism of acetone is disordered, the concentration of acetone increases, and the related disease is called acetonemia. Scientists have found that the concentration of acetone in diabetic plasma increases by two orders of magnitude. Even in the treated diabetic patients, the acetone concentration in plasma is much higher than that of normal plasma acetone. Some important enzymes such as propionyl-Co A carboxylase (EC 6.4.1.3) methylmalonyl-Co A mutase also cause an increase in acetone concentration in the blood . Recently, Kalapos pointed out in a review that acetone is an important disease marker molecule. Over the past few decades, scientists have developed methods to determine acetone in biological samples. Earliest, acetone in plasma was quantified using a colorimetric method. However, colorimetry has several disadvantages: lack of specificity and limited detection limits. In the last few decades, researchers have developed gas chromatography coupled with flame ionization detectors or mass spectrometer detectors to analyze the concentration of acetone in biological fluids and respiratory gases. Plasma acetone is derivatized with 2,4-dinitrophenylhydrazine (DNPH) and then analyzed by high performance liquid chromatography. It has been reported in the literature that solid adsorbents are used for adsorption and enrichment of acetone in human respiration and enriched in adsorbent acetone using gas chromatography coupled with mass spectrometry or other detectors for the quantitative analysis of acetone in respiration. Kundu et al. developed a very simple method to analyze acetone in the respiratory and urine samples. It is a dip-and-read color matching assay. However, the sensitivity of this method is poor and the analysis result has a large error. Recently, a simple new technique, SPME (Solid Phase Microextraction), was used to extract and enrich acetone in the respiratory, plasma, and bloodstreams. Then quantified in combination with GC-MS analysis [1]. The acetone in the plasma is evaporating from the plasma to the headspace of the sample under stirring, and then extracted by the solid phase microextraction extraction head. The extraction head is then desorbed at the gas chromatographic injection port and analyzed by gas chromatography-mass spectrometry [ twenty-three].
Diabetes is a chronic disease. With the action of insulin in the human body, glucose and starch can be converted into energy needed daily. Diabetes develops when insulin cannot be used effectively to produce insulin in the body. The cause of diabetes is not yet clear, but it is certainly related to genetic and environmental factors. Obesity and lack of exercise can easily lead to diabetes. Diabetes mainly includes four types, type 1 diabetes, type 2 diabetes, gestational diabetes and quasi-diabetes. Type 2 diabetes is the most common type. There are approximately 18 million diabetics in the United States, accounting for 6.3% of the population. In China, there are approximately 40 million diabetics. Diabetes is usually diagnosed by measuring the concentration of blood glucose. Measuring blood glucose is an invasive method, and Rooth et al. attempted to diagnose diabetes using a non-invasive method-breath gas analysis. Studies have shown that lipolysis in the body produces acetone, which is excreted by the blood and adsorbed by the breath. Since diabetics cannot produce insulin or insulin does not effectively convert glucose into energy, since glucose cannot be converted into energy in the body, only fat can be metabolized, resulting in a large amount of acetone in the blood and in the breath. In the plasma of diabetics, the acetone concentration increases by two orders of magnitude. Acetone in the breath of diabetic patients is also much higher than the acetone in the normal person's breathing gas [2]. Acetone has been recognized as a marker molecule for diabetes and ketoacidosis. Analyzing acetone in the breath has become a potential tool for the diagnosis of diabetes. Since breath analysis is a non-invasive method, it has been used to diagnose diseases. The development of respiratory gas analysis has great significance for the early diagnosis of diabetes. Maximilian et al. used chemical sensors to analyze respiratory gases for diagnosing disease, but chemical sensors had large quantitative errors. Gas chromatography is usually used for the analysis of respiratory gases. Since the amount of acetone and other compounds in the respiratory gas is in the nanomolar range, enrichment is required prior to GC analysis in order to increase the sensitivity and accuracy of the analysis. Chemical methods, adsorption, cold trapping, and membrane extraction techniques have been applied to the enrichment of respiratory gases. Recently, Pawliszyn et al. used solid phase microextraction to analyze acetone and other disease marker compounds in human respiratory gas and blood samples [3]. Due to the volatility and high activity of acetone molecules, it is difficult to accurately analyze the concentration of acetone in the breath. To overcome this problem, acetone was derivatized prior to gas chromatographic analysis. In this paper, we further developed solid-phase microextraction derivatization techniques for the analysis of acetone in the breath.

On-site, Rapid Analysis of Biochemical Signal Substances in a Miniature Portable Gas Chromatograph
The miniaturization of gas chromatography has become a hot topic. With the advent of commercial micro GCs, its application prospects will be very broad. At present, micro gas chromatography is mainly used in environmental analysis [4], and very few reports have been applied to biological analysis. At present, studies on chemical signal substances of plants such as methyl salicylate, methyl jasmonate, etc. are conducted in the laboratory. Under natural conditions, the defense response of plants to insect pests or viruses is studied, which is of theoretical and practical significance in phytochemical ecology. The rapid on-site analysis of phytochemical signal substances is imminent. The portable micro GC has become the first choice for on-site and rapid analysis of phytochemical signal substances. In 2002, Kunert et al. used a micro GC together with a sample pre-concentration device (Figure 5.1) to rapidly analyze the volatile compounds released from lima bean leaves and the compounds released from the aphid-infested peppers [5]. Figure 5.2 is a micro gas chromatogram of the compound released from leaves of lima beans induced with alamethici (ALA). Liu and Pawliszyn et al. used membrane extraction to enrich biomarker volatiles released from eucalyptus leaves and then carried out rapid on-site separation analysis using a portable gas chromatograph.
The analysis of human disease-related biochemical signal substances, especially human respiratory diseases, also requires on-site and rapid analysis. At present, scientists have developed some chemical sensors and applied them to the rapid analysis of respiratory gases. However, because chemical sensors are difficult to accurately quantify disease markers in respiratory gases, they are not suitable for the screening of diseases. As we all know, gas chromatograph is the best means for quantitative analysis of volatile compounds, and microchromatography has the characteristics of rapid on-site analysis, and may become an important tool for diagnosing diseases by analyzing disease markers in respiratory diseases. At present, although the application of microchromatography to analyze the molecular markers of disease in respiratory disease has not yet been reported. However, currently, humans have difficulties in early diagnosis of some major diseases such as cancer and diabetes, leading to difficult treatment and high costs. These requirements require the development of a simple and effective method for the screening of major diseases of diabetes and lung cancer. Rapid analysis of respiratory gas marker molecules has great promise for some major diseases.

Conclusion
The micro gas chromatograph that we designed and developed is due to the direct column heating method and the miniature detector (only two air sources: air and hydrogen are needed), and it has a light weight (<5kg). Therefore, it has the characteristics of on-site and rapid analysis. It is not only suitable for the analysis of harmful substances such as benzene series and formaldehyde in indoor air, but also can be applied to the analysis of biochemical signal substances. Combined with our method of measuring acetone in the breath, the micro GC is expected to become a new tool for screening diabetes, providing an ideal measure for early warning of diabetes. Based on the current research on lung cancer markers, we will carry out a large number of lung cancer blood volatiles analysis and lung cancer breath analysis to further confirm our findings; to search for lung cancer marker molecules in the breath, hoping to use microchromatography to analyze the breath The gas reaches the initial diagnosis of lung cancer. To exploit the characteristics of microchromatography, we will also conduct research on plant disease resistance chemical signals in the field, discover more disease-resistant chemical signal substances as soon as possible, and prepare these signal substances as ecological preparations for agricultural production.


Influence of the Tumor Microenvironment on Cancer Cells Metabolic Reprogramming

As with castles, tumor cells are fortified by surrounding non-malignant cells, such as cancer-associated fibroblasts, immune cells, but also nerve fibers and extracellular matrix. In most cancers, this fortification creates a considerable solid pressure which limits oxygen and nutrient delivery to the tumor cells and causes a hypoxic and nutritional stress. Consequently, tumor cells have to adapt their metabolism to survive and proliferate in this harsh microenvironment. To satisfy their need in energy and biomass, tumor cells develop new capacities to benefit from metabolites of the microenvironment, either by their uptake through the macropinocytosis process or through metabolite transporters, or by a cross-talk with stromal cells and capture of extracellular vesicles that are released by the neighboring cells. However, the microenvironments of primary tumor and metastatic niches differ tremendously in their cellular/acellular components and available nutrients. Therefore, cancer cells must develop a metabolic flexibility conferring on them the ability to satisfy their biomass and energetic demands at both primary and metastasis sites. In this review, we propose a brief overview of how proliferating cancer cells take advantage of their surrounding microenvironment to satisfy their high metabolic demand at both primary and metastasis sites.

inTRODUCTiOn Cellular heterogeneity of solid tumors strongly impacts tumor progression. This abundant heterocellularity drives the nature and abundance of the components of the extracellular matrix (ECM) and, for some cancers, makes tumor cells the minor cell type in terms of cellular amount. It appears that tumor cells take advantage of this dense microenvironment and are engaged in a complex dialog with their surrounding cells. A multitude of studies have emerged to dissect the inflammatory, metabolic, or oncogenic nature of the dialog between different cell types in tumors and have improved our knowledge of the various communication modes between cells; physical interactions, secreted molecules, extracellular vesicles (EVs), etc. In this review, we highlight how the stroma [mainly cancer-associated fibroblasts (CAFs)]-tumor cell metabolic axis increases the metabolic performance of tumor cells in addition to the cell's autonomous metabolic pathways. We also discuss how tumor cells recycle some metabolites, considered until now as "metabolic wastes, " to support their biosynthetic and bioenergetic needs. Finally, we point out the metabolic plasticity that metastatic tumor cells acquire to adapt to the microenvironments of both the primary and metastasis sites.

MeTABOLiC COMMUniCATiOn BeTween TUMOR CeLLS AnD THeiR neiGHBORinG CeLLS
Cancer-associated metabolic remodeling is not restricted to malignant cells but is also found in tumor-surrounding, nontransformed stromal cells. This stromal metabolic reprogramming is dictated by the tumor cells and, as a feedback loop, microenvironmental cells drive metabolic changes in tumor cells and/or provide metabolic resources required for tumor growth. CAFs are the most prominent cell type in the tumor microenvironment (TME) (1, 2) and have emerged as key components of the stromal-epithelial metabolic coupling. In pancreatic cancer cells, Sherman et al. have shown through a transcriptomic approach that soluble cues from patient-derived CAFs induce deep metabolic alterations that are similar to those driven by K-RAS. The metabolic pathways found the most enriched in stroma-activated tumor cells are associated with steroid and unsaturated fatty acid biosynthesis, and also with glycolysis and gluconeogenesis.
Moreover, stromal cues increase flux through glycolysis and pentose phosphate pathway (PPP) and enhance tricarboxylic acid (TCA) cycle intermediates. Hence, the stroma induces genomic and metabolic responses that strengthen pancreatic tumor progression (3). A recent in situ study analyzing single cell enzymatic activities in intact tumor tissues showed that CAFs have a higher glycolytic activity than the different subtypes of breast tumor cells (4). CAFs metabolize glucose through anaerobic glycolysis and export lactate which is then taken up by oxidative cancer cells to increase their tumorigenic potential (5), a phenomenon coined the "reverse Warburg effect" which is also reported in other cancers (6) (Figure 1). The CAFs's-enhanced glycolysis is induced by cancer cells in response to hypoxic or oxidative stress injuries (7,8) and is not a general adaptive metabolic feature of CAFs. Indeed, even if pancreatic-CAFs are prone to glycolysis (9), the lactate is secreted at a lower rate than alanine, a non-essential amino acid (NEAA) (10). Secretion of alanine by CAFs results from autophagy-induced protein breakdown in these cells and, once taken up by cancer cells, it has an unusual metabolic rate. Instead of contributing to protein synthesis, alanine is converted into pyruvate in the mitochondria to provide energy and lipids essential to pancreatic-ductal adenocarcinoma (PDAC)-cell survival and growth. In breast tumors, CAFs release kynurenine, a tumor-promoting metabolite arising from tryptophan breakdown, whose synthesis is increased in response to tumor-derived lipid mediators (prostaglandin E2) (11). In these tumors, transcriptional profiles of caveolin-1-deficient fibroblasts reveal an over-expression of ketogenic genes promoting ketone bodies (KB) production. Hence, CAFs contain a pool of KB that can be used as an energy source by oxidative breast tumor cells, and that can promote breast tumor growth (Figure 1) (12). CAFs also mediate tumor cells' metabolic reprogramming in a paracrine manner through diffusible or EV molecules. CAF-derived EVs promote a metabolic switch from mitochondrial oxidative phosphorylation (OXPHOS) to aerobic glycolysis to satisfy prostate and pancreatic tumor cell needs in ATP (Figure 1). Hence, EVs increase reductive glutamine's contribution to lipogenic acetyl-CoA cycle and the use of extracellular acetate as an additional carbon source for fatty acid synthesis. These metabolic changes are induced by intra-EV metabolites and miRNA-targeting OXPHOS genes (13). CAF-secreted cytokines, such as hepatocyte growth factor, have also been reported to favor glucose uptake in human breast tumor cells by increasing glucose transporter 1 levels (14) (Figure 1).
In tumors, nutrient-deprived conditions strengthen the stromal-epithelial metabolic shares. Under glutamine restriction, patient-derived CAFs survive and undergo a metabolic reprogramming leading to an increase in glutamine synthesis. Enhanced glucose entry into the TCA cycle, along with amino acid (AA) and lactate intake, contribute to the carbon supply for glutamine synthesis, while the nitrogen donors are branchedchain amino acids, NEAA, and ammonia. Once secreted and taken up by ovarian cancer cells, glutamine rescues tumor growth by increasing expression of genes involved in cell cycle, fatty acid, and nucleotide synthesis (15). Under AA and pyruvate starvation, CAFs enhance the packaging of cargo metabolites into EVs to rescue prostate and pancreatic tumor cell survival (13). As metabolites present in CAF-EVs are in their active form, their impact on pancreatic cell proliferation is instantaneous and strong (e.g., exosomal metabolites contribute to one-third of the TCA cycle flux), but short term (16). Interestingly, the EV-metabolite supply is K-RAS-independent (13) suggesting that CAF-mediated metabolic remodeling may be a common trait of cancers.
In addition to metabolic dialog with stromal cells, tumors cells exchange diverse metabolites between one another, a phenomenon initially referred to as metabolic symbiosis. One of the most described processes of metabolic symbiosis relies on the potential of malignant cells to increase their glucose consumption and produce large amounts of lactate through aerobic glycolysis (the Warburg effect). In hypoxic tumors, such as PDAC, large amounts of lactate secreted by highly glycolytic hypoxic cells are taken up by normoxic neighboring tumor cells and promote their proliferation. Lactate thereby contributes to the metabolic symbiosis between both hypoxic and normoxic cellular compartments of the tumor (17) (Figure 1). In non-small-cell lung cancers, Faubert et al. showed that tumor cells also metabolize lactate, proving lactate's contribution as a fuel of the TCA cycle exceeds that of glucose in vivo (18). Treatment of pancreatic neuroendocrine, breast and renal cancers with angiogenic inhibitors induces regionalization of these tumors into hypoxic and normoxic zones and force normoxic cells, in close proximity to hypoxic ones, to operate a metabolic symbiotic shift toward the use of lactate diffusing from hypoxic regions. Interestingly, lactate catabolism by normoxic tumor cells is branched to the glutamine metabolism through lactate-derived pyruvate transamination. This reaction allows the production of alanine and α-ketoglutarate, thereby fueling the TCA cycle (Figure 1). This symbiotic metabolism is dependent on mTOR signaling as treatments of tumors with a combination of angiogenic inhibitors and the mTOR inhibitor rapamycin suppresses lactate catabolism by normoxic cells. Normoxic cells switch their metabolism back to a more glycolytic phenotype to the detriment of hypoxic cells which are then devoid of bio-available glucose (19).
Interestingly, lactate not only participates in metabolic processes but also contributes to oncogenic signaling pathways. This function is not restricted to lactate as many by-products of metabolic pathways act as signaling molecules (20). This notion of the dual function of metabolites, being involved in metabolic and oncogenic signaling, strengthens the tight association between tumor metabolism, cell cycle dysregulation, and aberrant cell proliferation. Interestingly, these processes are directed by the circadian clock which governs biological rhythms for tissue homeostasis (21,22). Hence, an integrated and dynamic view of how metabolic pathways and cell cycle machinery interact with the circadian clock in the context of tumor progression deserves to be better explored, and would help to develop efficient metabolic therapeutic strategies as circadian timing of drug administration impacts both the efficacy and the toxicities of most pharmacotherapies (23).

MeTABOLiC ReCYCLinG
Increased hetero-cellularity of the TME is associated with substantial ECM deposition. This densification of the ECM during tumor growth is associated with changes in stiffness, elasticity, and mechanical properties of the microenvironment but also increases the richness of macromolecules surrounding the tumor cells. Hence, the TME becomes a nutrient supply center for the tumor cells as it is composed of abundant macromolecules, such as collagen, hyaluronan, fibronectin, albumin, lipids, etc. To take advantage of this enriched microenvironment and optimize the use of the macromolecules, tumor cells use macropinocytosis, a non-selective endocytic process, to take up extracellular components and internalize them into vesicles. Products derived from successive degradation of the vesicles' contents are released in the tumor cells' cytosol as ready-to-use nutrients. Macropinocytosis occurs in several types of cancers and is largely described as a central process of acquisition of nutrients by PDAC cells. In vivo, inhibition of macropinocytosis impedes the growth of subcutaneous xenografted pancreatic tumors (24). Vander Heiden's lab also demonstrated that PDAC satisfies its avidity for macromolecules such as albumin using macropinocytosis and locally increases its breakdown to derive pools of AAs (25). In this context, K-RAS appears to be a main oncogenic driver of this process. In vitro, K-RAS mutant PDAC cells compensate for a lack in AAs in culture medium and maintain a high proliferative rate due to their capacity to recover the needed AAs pools from scavenged and catabolized albumin (26). Interestingly, the ability of spontaneous PDAC to take up macromolecules is not only restricted to albumin but also includes fibronectin. Recently, we also showed that PDAC cells, when deprived of nutrients, scavenge environmental collagen which appears essential for their survival especially under glucose deprivation, the condition in which macropinocytosis is activated. Subsequent digestion of collagen supplies PDAC cells with pools of proline, a main component of collagen molecules. Proline catabolism by PRODH1 enriches the cells with TCA intermediates, especially under nutrient deprivation, and appears to be a promoting metabolic pathway of PDAC growth (Figure 1) (27).
Proliferative tumor cells can also recycle metabolites found in the circulation, such as acetate and KB. Both have been described to be carbon sources to supply the tumor with energy and biomass. Breast cancer cell lines consume large amount of acetate when subjected to metabolic stress and hypoxia to synthesize fatty acids and supply the membrane with phospholipids. Acyl-CoA short chain synthetase 2 promotes the acetate uptake and produces the acetate-derived acetyl-CoA pool needed for fatty acids synthesis (28). KB represented by acetone, acetoacetate, and β-hydroxybutyrate (β-OHB) constitute another family of metabolites that are usable by tumor cells. In physiological conditions, KB are produced in liver, released into the circulation and reach tissues/organs to supply cells in biomass and energy especially in low glucose condition. KB catabolism (ketolysis) consists in oxidation of β-OHB, the most abundant KB in the circulation, to form acetoacetate which is converted by 3-oxoacid CoA-transferase 1 (OXCT1) to produce successively acetoacetyl-CoA and 2 molecules of acetyl-CoA. The latter directly fuels the TCA cycle and supplies cells with ATP. In human hepatocellular carcinoma (HCC), tumor cells overexpress OXCT1 when nutrient-deprived, suggesting that these cells, contrary to the normal hepatocytes counter-part which synthesize and produce KB, catabolize β-OHB when they are nutrient stressed. Indeed, OXCT1 favors β-OHB uptake by HCC cells which use β-OHB to fuel their TCA cycle and produce ATP to promote their proliferation (Figure 1). Moreover, OXCT1 expression in HCC cells is dependent on the mTORC2-AKT-SP1 signaling axis and induction of ketolysis by OXCT1, by supplying cells with ATP, suppresses AMPK activation upon nutrient starvation, avoiding the deleterious excessive autophagy and promoting HCC cell survival and proliferation (29). In melanoma and leukemia driven by the BRAF-V600E mutation, acetoacetate levels are increased. In tumor cells, acetoacetate enhances BRAF-V600E binding to MEK1 and thereby activates the MEK-ERK signaling axis and contributes to BRAFV600E tumor growth (30,31). In breast cancer, tumor cells, by increasing the number of MCT2 transporters at the plasmic membrane, are also able to take up β-OHB produced by adipocytes localized at the tumor site. This uptake favors the clonogenic potential of MCT2 positive tumor cells as well as their capacity to form a tumor mass in vivo (32).
Ammonia recycling by tumor cells is another example of how cancer cells benefit from the capture of metabolites released either into the TME by neighboring cells or in to the circulation by the liver. Ammonia produced during the conversion of glutamine to glutamate was, until recently, considered as waste. In a metabolic symbiosis context, Spinelli et al. recently demonstrated that the recycling of ammonia maximizes nitrogen utilization by glutamate dehydrogenase for glutamate synthesis from α-ketoglutarate. Glutamate then contributes to proline or aspartate synthesis. Importantly, the authors showed that this recycling accelerates proliferation of breast tumors (33). Hence, ammonia and KB recycling by tumors highlights the need to consider the interactions, in the pathophysiologic context of cancer, between tumors and the rest of the host organism. It is then crucial to integrate the metabolites exchange between the tumor and metabolic organs/tissues of the host in ex vivo studies of the metabolic reprogramming of tumors. Although the set up of in vitro and in vivo experimental models to consider such metabolic communication appears ambitious and complex, it emerges nowadays as a necessity to take tumor metabolism studies to a new level and improve the relevance of translational studies for effective metabolic therapies (34). In line with this, mathematic models of the tumor-organs metabolic interactions become essential tools in establishing experimental models.

MeTABOLiC FLeXiBiLiTY OF MeTASTATiC CeLLS
The metastasis sites' microenvironment retains some similarity to the primary site for components such as collagens, hyaluronan, and smooth muscle actin-expressing cells (35), but also differs from the primary microenvironment in the level of their cellular/ acellular components and available nutrients. Hence, tumor cells must develop a metabolic plasticity to satisfy their biomass and energetic demands required for their proliferation in both primary and metastasis sites. It is, therefore, important to highlight a few studies revealing the metabolic programs of metastatic cells according to their tissue of origin and/or to the metastasis site microenvironment. Christen and collaborators revealed that lung interstitial fluid has increased levels of pyruvate compared to blood plasma resulting in higher pyruvate levels in lung metastases compared to primary breast tumors. Consequently metastatic breast cancer cells take advantage of the pyruvate availability in their metastatic environment by increasing pyruvate carboxylasedependent anapleurosis compared to primary tumor cells (36) (Figure 2). As a metabolic organ, the liver produces a plethora of metabolites usable by colonizing metastatic tumor cells, creatine being one of them. Creatine is phosphorylated by creatine kinase (CK), resulting in phosphocreatine, useful to shuttle high energetic phosphate when it enters into cells to produce ATP from ADP when energetic needs exceed ATP synthesis. When colon cancer cells metastasize in the liver, they must face the new environmental hypoxic conditions. Secretion of the brain-type CK (CKB) by colon metastatic cells into the extracellular space of the liver allows production of phosphocreatine from extracellular ATP and creatine. Phosphocreatine uptake by liver-disseminated metastatic cells supplies them with ATP to survive the hypoxic microenvironment (Figure 2). Consequently, inhibition of CKB in disseminated colon cancer cells impedes metastatic Example of metabolic pathways activated in breast, colon, and pancreatic metastatic cells during colonization of lung, liver, and liver/lung, respectively. Abbreviations: oxPPP, oxidative branch of the pentose phosphate pathway; CKB, brain-type creatine kinase.
colonization of the liver. Inhibition of CKB and the creatine transporter in PDAC cells also limits their metastatic potential, highlighting that a common metabolic targeting of disseminated cells originating from different gastrointestinal organs is feasible to abolish metastatic progression (37). Interestingly, during the evolution of localized PDAC toward a metastatic disease, a widespread epigenetic reprogramming occurs. Moreover, metastasis at distant sites appears to be dependent on the oxidative branch of the PPP (oxPPP). Indeed, 6-phosphogluconate dehydrogenase, the enzyme responsible for nucleotide synthesis through the oxPPP, not only controls tumor growth at metastasis sites but also governs chromatin reprogramming as well as malignant gene expression. This illustrates that epigenetic events leading metastatic PDAC progression are dependent on the metabolic reprogramming of metastatic cells (38) (Figure 2).
In metastasis sites, tumor cells also influence the CAFs' metabolic activity. Indeed, breast cancer cells transfer miR-122 through EVs to resident fibroblasts in lung pre-metastatic niches. By reducing glucose consumption by fibroblasts, miR-122 increases glucose availability to tumor cells (39). Hence, metabolic remodeling of stromal cells in metastasis sites provides favorable "soil" for seeding and growth of cancer cells.

COnCLUSiOn
Metabolic cooperation between the TME and cancer cells contributes to tumor growth, especially in nutrient-or oxygen-deprived microenvironments. As a consequence, targeting the metabolism of stromal cells impedes tumor progression to the same extent as targeting the tumor cells' metabolic mediators. Therefore, when considering metabolic targeting of tumors as an anti-cancer therapy, targeting only the cancer cell autonomous metabolism would not be sufficient. Moreover, we highlight in this review that the nurturing microenvironment supplies tumor cells with macromolecules or metabolites and fuels metabolic pathways especially in stressful conditions. Fortunately, mechanisms used by tumor cells for the uptake of such environmental nutrients are being progressively uncovered. Development of therapeutic metabolic approaches must, therefore, take into account that the metabolic reprogramming of tumor cells is flexible and evolves along with microenvironmental changes. As such, a unique metabolic therapeutic window is not conceivable. This highlights the need to develop combined metabolic targeting to circumvent tumor metabolic plasticity and abolish tumor progression in the long term.

AUTHOR COnTRiBUTiOnS
VG, FG, and SV conceived, organized, and wrote the manuscript.

ACKnOwLeDGMenTS
We thank Kendra Delicaet for editing of the manuscript.

FUnDinG
This work was supported by a French National League against Cancer grant, a French Association for Cancer Research "ARC" grant, and a National Institute of Cancer "INCa" grant (grant 2015-115).