extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Expression Profiles of SnoN in Normal and Cancerous Human Tissues Support Its Tumor Suppressor Role in Human Cancer

SnoN is a negative regulator of TGF-β signaling and also an activator of the tumor suppressor p53 in response to cellular stress. Its role in human cancer is complex and controversial with both pro-oncogenic and anti-oncogenic activities reported. To clarify its role in human cancer and provide clinical relevance to its signaling activities, we examined SnoN expression in normal and cancerous human esophageal, ovarian, pancreatic and breast tissues. In normal tissues, SnoN is expressed in both the epithelium and the surrounding stroma at a moderate level and is predominantly cytoplasmic. SnoN levels in all tumor epithelia examined are lower than or similar to that in the matched normal samples, consistent with its anti-tumorigenic activity in epithelial cells. In contrast, SnoN expression in the stroma is highly upregulated in the infiltrating inflammatory cells in high-grade esophageal and ovarian tumor samples, suggesting that SnoN may potentially promote malignant progression through modulating the tumor microenvironment in these tumor types. The overall levels of SnoN expression in these cancer tissues do not correlate with the p53 status. However, in human cancer cell lines with amplification of the snoN gene, a strong correlation between increased SnoN copy number and inactivation of p53 was detected, suggesting that the tumor suppressor SnoN-p53 pathway must be inactivated, either through downregulation of SnoN or inactivation of p53, in order to allow cancer cell to proliferate and survive. These data strongly suggest that SnoN can function as a tumor suppressor at early stages of tumorigenesis in human cancer tissues.

Introduction
SnoN (Ski-novel protein) was fist discovered as an oncoprotein that can induce transformation of chicken and quail embryonic fibroblasts when overexpressed [1][2][3][4]. In mammals, SnoN contains both pro-oncogenic and anti-oncogenic activities. The pro-oncogenic activity of SnoN is dependent on its ability to bind to the Smad proteins and antagonize the cytostatic responses of TGF-b [5,6]. Suppporting its pro-oncogenic role, SnoN expression is elevated in most human cancer cell lines, and reduction of SnoN expression by shRNA in invasive breast cancer and lung cancer cell lines inhibits tumor growth both in vitro and in vivo [7]. More importantly, overexpression of SnoN in the mouse mammary gland accelerates breast cancer development and pulmonary metastasis induced by the Polyoma middle T antigen (PyVmT) [8], providing the first in vivo support for the prooncogenic activity of SnoN. In recent years, however, more and more evidence have emerged indicating that SnoN can also act as a tumor suppressor. Heterozygous mice lacking one copy of the snoN gene show an increased susceptibility to carcinogen-induced tumorigenesis, suggesting that SnoN may protect mice from carcinogenesis [9]. We have recently shown that high levels of SnoN inhibit oncogene-induced transformation of primary mouse embryonic fibroblasts in vitro and block tumor development in vivo in a two-step skin carcinogenesis mouse model [10]. This antioncogenic activity of SnoN is independent of its ability to repress TGF-b signaling and likely stems from its ability to induce stabilization of p53. In response to cellular stress, high levels of SnoN is recruited to the PML nulcear bodies where it induces stabilization of p53, leading to increased cellular senescence and apoptosis [10]. Thus, high levels of SnoN in cells may activate the PML-p53 tumor suppressor pathway to inhibit tumor growth. Finally, SnoN may also inhibit tumor metastasis since reducing SnoN expression enhances epithelial to mesenchymal transition (EMT) of lung and breast cancer cells in vitro and tumor metastasis in vivo [7].
Given the complex roles of SnoN in tumorigenesis, it is important to determine its expression pattern in normal human tissues and monitor how it changes during malignant progression. SnoN is ubiquitously expressed in all embryonic and adult tissues. Its expression is upregulated during specific stages of embryogenesis and organ morphogenesis [11]. In untransformed cells and normal tissues, SnoN can be detected in both cytoplasm and nucleus, whereas in cancer cell lines, SnoN is largely nuclear [8,12,13]. In addition, SnoN expression can be regulated at the level of gene amplification, transcriptional activation and protein stability. The human snoN gene is located at chromosome 3q26.2, a locus frequently amplified in many cancer types, including that of esophagus, ovary and many others [5]. In agreement with this, SnoN expression is upregulated in many cancer cell lines [6]. However, whether SnoN expression is also increased in human cancer tissues remains controversial. A number of studies have examined this issue [13][14][15][16][17][18], but no consistent pattern has emerged. While some studies reported an increase in SnoN RNA and protein levels in some cancer tissues and that this higher SnoN expression correlated with poor differentiation, deeper invasion and poor patient survival [13,14,16], others detected a decrease in SnoN expression in similar cancers, particularly in dysplastic and highly invasive cancers [15,17,18]. The potential relationship between localization of SnoN and malignant status of cancer also varied from study to study. These conflicting data suggest a more complex relationship between SnoN expression and malignant transformation and justify the need for a broader examination of SnoN expression in normal and cancer tissues of different malignant stages.
In this report, we examined SnoN expression in four types of normal human tissues and matching cancer tissues of various clinical stages of malignancy to assess whether alterations of SnoN expression correlate with tumor malignancy and/or status of p53 inactivation. These four types, esophageal, ovarian, breast and pancreatic tissues, were chosen because SnoN has been implicated in the development of these cancers and/or in the normal functions of these tissues. Esophageal and ovarian cancers often involve amplification of the 3q26 amplicon, and the copy number of the SnoN gene as well as SnoN transcript levels have been found to increase in these cancers [14,16,19,20]. However the expression pattern of SnoN protein in normal ovary and in ovarian adenocarcinoma have not been defined. Pancreatic cancer is closely associated with the TGF-b/Smad pathway, and Smad4 is inactivated in nearly 60% of pancreatic cancer [21]. As a negative regulator of Smad4, SnoN may also play a role in the development of pancreatic cancer. Finally, our recent studies using various mouse models have shown that SnoN expression is dynamically regulated during mammary gland development [8] and is implicated in the malignant progression of breast cancer [7,8,12]. By examining the location and levels of SnoN expression in these tissues, we hope to gain a better understanding of the functions that SnoN play during tumorigenesis in both the epithelium and stromal environment and how this may correlate with p53 inactivation.

Normal and Cancer Tissue Arrays
All arrays were purchased from Biomax (Cybrdi Inc). These arrays come with information on clinical stages, pathology grades and TNM. The esophageal array contains pairs of tumor biopsy samples and matched normal samples from 39 patients with histologically and clinically classified adenocarcinoma: 8 patients with grade I, 19 patients with grade II, and 11 patients with grade III tumors.
The ovarian tumor array contains tumor biopsy samples and 10 normal matched samples from 69 women aged 50+/210. The tumors were classified to be ovarian adenocarcinomas: 3 patients with ovarian clear carcinoma, 12 patients with grade I, 29 patients with grade II, and 25 patients with grade III adenocarcinomas (all with lymph node metastasis but not distant metastasis).
The pancreatic tumor array contains samples from 90 patients and five normal pancreatic tissue samples . The tumor types on this  array include ductal adenocarcinoma, mucinous adenocarcinoma,  neuroendocrine carcinoma, adenosquamous carcinoma, carcinoid, and solid pseudo-papillary carcinoma. They have been  classified into different clinical classes based on histological  evaluation: 21 patients with grade I, 59 patients with grade II, 8 patients with grade III (many with lymph node metastasis), and 2 patients with grade IV tumor with distant metastasis.
The breast cancer array contains duplicated tumor biopsy samples from 31 patients, with six normal samples from six matching patients, and 9 metastatic samples from 9 matching patients. These tumors were clinically classified by histological analysis to be infiltrating ductal carcinoma: 9 patients with grade IIA, 4 patients with grade IIB, 10 patients with grade IIIA, 5 patients with grade IIIC, 9 matching patients with lymph node metastatic, and 1 patient with metastatic adenocarcinoma to the ovary. Because no difference in SnoN expression was detected between grade IIA and IIB or between grade IIIA and IIIC tumors, we grouped these samples together as grade II or grade III tumors. In addition, 24 pairs of breast ductal carcinoma in situ (DCIS) samples with matching normal tissues were obtained from UCSF cancer center.
The thickness of all the arrays is 5 mm. The breast cancer array contains duplicate samples from each patient and esophageal, ovarian and pancreatic arrays have one section from each patient.

Immunohistochemistry
For SnoN immuostaining. Paraffin-embedded arrays were deparaffinized in xylenes then rehydrated in a series of ethanol gradient. Sections were immersed in 0.85% Nacl for 5 minutes, then in PBS for 5 minutes, and fixed with 4% paraformaldehyde (PFA). Permeabilization of the slides was carried out with 20 mg/ ml proteinase K for 10 minutes at room temperature (RT), followed by washing in PBS for 5 minutes. Samples were fixed a second time with 4% PFA, and blocked overnight in 1%BSA/ 10% newborn calf serum/0.02% Triton X-100 in PBS in a humidified chamber at 4uC. The staining was performed with an anti-SnoN antibody recognizing a C-terminal peptide of SnoN [12] at a concentration of 1 mg/ml for 2 hours along with a peptide competition control and a negative control without primary antibody. After 3 washes with IF buffer (10% newborn calf serum/ 0.02% Triton X-100 in PBS) for 15 minutes each, fluorescent conjugated secondary antibody (Alex488 anti-rabbit antibody 1:400 dilution) was added to the slides for 1 hour at RT in the dark. Slides were washed once with IF buffer for 15 minutes and twice with PBS for 10 minutes each, followed by addition of one drop of Vectashield DAPI mounting medium (H-1200; Vector Laboratories, Burlingame, CA).
The sections from the same tissue types (normal and cancerous) were all stained in the same experiment under identical conditions, and images were taken under the confocal microscope in one session. In addition, the same antibody lot and dilutions were used for all stainings described in this paper. Under no circumstances was the antibodies re-used in any of the experiments.
For p53 immunohistochemistry. Paraffin-embedded sections were deparaffinized in xylenes, rehydrated in ethanol, and incubated with 3% H 2 O 2 for 10 minutes to quench endogenous peroxidase activity. Then sections were microwaved at 95uC for 30 minutes in sodium citrate buffer (pH 6) [22] and blocked using the Tyramide Signal Amplification Biotin System Kit (Perkin Elmer, Boston, MA) for 30 minutes at RT. Slides were treated for 30 minutes with anti-p53 at 1:200 dilution (DO-7 clone; Abcam, Cambridge, UK). After washing 365 min in 0.05% TritonX in PBS, slides were incubated in biotinylated secondary antibody for 30 min at RT. All subsequent steps were performed per manufacturer's protocol. For visualization, DAB was used as the peroxidase substrate (SK-4105; Vector Laboratories, Burlingame, CA).

Image Capture and Semi-Quantitative Data Analysis
For each SnoN-stained tumor sample, at least three images were captured using the LSM 510 confocal microscope under the 20x objective to make sure that the major areas for each sample are included in the quantification. SnoN intensity (the number of pixels/ area) of each of the three areas was calculated using the image J software, and the size of the area was kept constant throughout the quantification process. The numbers were then averaged for each tumor sample. The negative control and peptide competition background signals were substracted from all the samples during quantification. The p53 images are taken with the Zeiss AxioImager fluorescence microscope. Nuclear p53 signal intensity was quantified in a 0 (negative) to 5 (highly intense staining) scale.
For analysis of potential association of TP53 mutation and SKIL amplification, we performed data mining of the Novartis cell line encyclopedia (CLE) that contains 947 human cancer cell lines [23], in which copy numbers and mutations of SnoN (SKIL) and p53 genes have been characterized using Affymetrix SNP6 microarray or RNAseq. We analyzed 914 cell lines by separating the cell lines based on the frequency of SnoN amplification and examining the frequency of TP53 mutation within each group.
Statistical analysis was performed using the student's t-test function in bioconductor R package as 2-tailed t-tests assuming unequal variances. Pearson's correlation coefficient was calculated using the R package, and the significance of the observed correlation coefficient was determined using one-sample t-test against the population of correlation coefficients in-between p53 and all Entrez genes in the human genome. Potential correlation between SnoN expression and p53 inactivation in cancer tissues was evaluated using the Kruskal-Wallis test.

SnoN is Expressed in Normal Mammalian Tissues
To determine a normal pattern of SnoN in various human tissues and establish a baseline for further analysis of cancer tissues, we first examined SnoN expression in 39 normal esophageal tissue samples, 10 normal breast samples, 6 normal ovarian and 5 normal pancreas samples by immunohistochemistry with anti-SnoN. This affinity-purified antibody was raised against a Cterminal peptide of human SnoN and has been previously characterized for imunofluorescence and immunohistochemistry studies [8,12].
The mucosa of the esophagus contains three layers: the nonkeratinized stratified squamous epithelium, the lamina propria consisting of loose connective tissue, and the muscularis mucosa consisting of smooth muscle. In the esophageal stratified squamous epithelium, SnoN was expressed at moderate to high levels in the cytoplasm, with a stronger staining in the upper surface part of the flattened epithelium than in the lower layer of cuboidal cells ( Figure 1A). In the lamina propria, SnoN was predominantly localized in the cytoplasm of cells in the connective tissue, but can also be detected in the nucleus of some fibroblasts. Blood vessels expressed SnoN at a high level, and the skeletal muscle of the muscularis mucosa had the highest SnoN staining ( Figure 1A). Thus, SnoN is expressed in both epithelial cells and the stromal compartment, and given its high levels of expression in muscles, blood vessels and fibroblasts, it may play an important role in regulating the stromal microenvironment.
In the normal ovarian tissue, SnoN was predominantly cytoplasmic and expressed at moderate levels in the primordial and primary epithelial follicle cells and stromal cells ( Figure 1B). In the normal pancreas, SnoN was present in the cytoplasm of the epithelial cells of the acini and intralobular ducts at moderate levels, and in the lobular connective tissue septa at a lower level ( Figure 1C). Finally, in normal breast tissues, SnoN was expressed in the cytoplasm of epithelial cells of the lobuli and terminal ducts ( Figure 1D), similar to what has been reported before [8,12]. It was also detected in the stroma at a lower level. Thus, SnoN is present in all the epithelial and stromal layers of these four normal tissue types at varying levels. In the epithelial cells of all tissue types, SnoN is predominantly localized in the cytoplasm. This is consistent with our earlier findings [8,12].

SnoN Expression is Reduced in Lower Grade Esophageal Adenocarcinoma but Comparable to that in WT Tissues in the High Grade Tumor Stroma
We next examined the SnoN expression levels and patterns in 39 esophageal adencarcinoma tissue samples by immunofluorescence staining followed by confocal imaging at both 20X and 40X magnification and compared them with that in the matching normal samples. These esophageal adenocarcinoma samples have been classified clinically from grade I to III. Notably, the multilayered tissue structure seen in normal esophageal tissue has been lost completely in these adenocarcinoma samples, and tumor stromal cells are interspersed with tumor epithelium. Our semiquantitative analysis showed that compared to the normal squamous epithelium, SnoN expression was significantly reduced in grade I (p = 0.00016) and to a lesser extent, grade II tumors (p = 0.142) (Figure 2A and 2B). Interestingly, as tumors progressed to a more malignant stage (grade III), SnoN expression recovered gradually to a level comparable to or higher than that in normal controls (two representative samples, one similar to normal and one higher are shown in Figure 2A). SnoN expression in the stromal cells and connective tissue in grade I and II tumors were also decreased significantly from that in normal tissues. Grade III tumor stroma, however, showed a significant upregulation of SnoN expression than grade I tumors (p = 0.0068), reaching a level similar to and in 55% of the samples, higher than that in normal tissues (last panel, Figure 2A), especially in infiltrating fibroblasts and inflammatory cells (lymphocytes, leukocytes and phagocytes) (Figure 2A and 2C). However, due to the large variations among individual grade III samples, the mean change in stroma SnoN levels over that in WT tissues was not statistically significant. Notably, SnoN remained largely cytoplasmic in these tumor samples. Our data suggest that SnoN expression is downregulated in patients with low grade esophageal adenocarcinoma but its expression is re-established in high grade tumors, in particularly in the stroma (for detailed numbers, see Table 1). This suggests that SnoN expression may vary at different stages of malignancy and that SnoN may play different roles at different stages of tumorigenesis.

SnoN Expression is Reduced in Lower Grade Ovarian Adenocarcinoma Tissues but Elevated in High Grade Tumor Stroma
Similar to what have been observed in esophageal cancer, SnoN expression was downregulated in grade I and to a lesser degree, grade II ovarian adenocarcinoma samples in both tumor epithelia and stroma (Figure 3 and Table 1), but recovered again in grade III tumors. Grade III ovarian adenocarcinoma samples showed heterogeneity in SnoN expression, with 60% of the samples displaying a higher SnoN staining (last panel, Figure 3A) than the normal tissues and others exhibiting a weaker SnoN staining (panel #4, Figure 3A). As a result of this heterogeneity, statistical analysis showed that there was no overall difference in SnoN expression between normal ovarian tissue and grade III tumor samples. However, when compared to the lower grade tumor samples, the stromal compartment of the grade III tumor samples showed a much stronger SnoN staining ( Figure 3A and 3C). These stromal cells were identified by pathologists as infiltrating fibroblasts and inflammatory cells. Again, SnoN remained largely cytoplasmic in these tumor samples. Thus, similar to that has been observed with esophageal cancer, SnoN expression in ovarian cancer also appeared to be lower in early stages of malignant progression.

SnoN Expression is Decreased in Pancreatic Adenocarcinoma Samples
The pancreatic cancer tissue array contains multiple types of adenocarcinoma of all three clinical grades (I-III), including ductal adenocalarcinoma, mucinous adenocarcinoma, neuroendocrine carcinoma, adenosquamous carcinoma, carcinoid, and solid pseudo-papillary carcinoma. In all pancreatic cancer samples across all three tumor grades, SnoN was cytoplasmic and was expressed at a lower level than that in the normal age-matched samples ( Figure 4A-4B and Table 1). Across the tumor grades, SnoN expression was the highest in stage III pancreatic adenocarcinoma ( Figure 4B). However this increase was not above the normal tissue controls. The tumor stromal compartment did not have a higher SnoN staining than the normal stroma ( Figure 4A and 4C). Thus in none of the tumor tissues was SnoN expressed at a higher level than that in normal tissues, consistent with a tumor suppressor role of SnoN in pancreatic cancer.

SnoN Expression is Significantly Reduced in Breast Ductal Adenocarcinoma
The breast cancer tissue array contains duplicated biopsy samples from patients with infiltrating breast ductal carcinoma of  Table 1). On the contrary, SnoN levels were significantly lower than the control samples and no correlation was seen between its expression levels and the presence of lymph node metastases ( Figure 5B). SnoN was expressed at high levels in very few samples and was predominantly cytoplasmic. For most of the tumor samples, SnoN expression in the stroma was markedly lower than that in the normal control samples ( Figure 5A and 5C).  To test the possibility that SnoN may be elevated during early stages of tumorigenesis but reduced at late stage of malignant progression, we collected and stained 24 human DCIS samples and their matching normal tissues for SnoN expression. Interestingly, although in any individual cell in the DCIS tissues, the level of SnoN expression was similar to or in a few cases, lower than that in cells in normal tissues, due to a markedly increased epithelial content in DCIS samples, the overall SnoN level in the whole tissue was much higher than that in normal tissues ( Figure 5D). Thus, the overall increase in SnoN level in DCIS samples reflects the significantly elevated epithelial contents in these tissues but not the upregulation of SnoN expression in individual cells.
Taken together, unlike previously proposed, SnoN expression is reduced in lower grade malignant tumors of esophagus, breast, pancreas and ovary (Table 1). In esophageal and ovarian cancer, and to a lesser degree in pancreatic cancer, SnoN expression gradually recovered in grade III tumors. This finding is consistent with the model that SnoN may function initially as a tumor suppressor in malignant progression, possibly through activating p53. The model further predicts that in order for tumor cells to overcome the tumor suppressive activity of the SnoN-p53 pathway, they have to either inactivate p53 or delete SnoN.

Elevated SnoN Expression Correlates with Inactivation of p53 in Human Cancer Cell Lines but not in Primary Tumor Tissues
In contrast to its reduced expression in primary tumor samples, SnoN have been shown previously to be elevated in esophageal, ovarian and breast cancer cell lines [7,16,19,20], due possibly to gene amplification and/or transcriptional activation. Since this elevated SnoN can activate p53 to trigger senescence, these cancer cells are predicted to inactivate this p53-dependent pathway in order to sustain proliferation. We therefore tested the hypothesis that cancer cells with elevated SnoN may also show inactivation of p53. We analyzed 914 cancer cell lines in the Novartis cell line encyclopedia (CLE) [23], in which copy numbers and mutations of SnoN and p53 genes were characterized using Affymetrix SNP6 microarray or RNAseq technology, to determine whether samples with increased copy number of the SnoN gene also tend to show inactivation of p53 as indicated by a loss of p53 copy number or the presence of known inactivating mutations. Based on the mutation status of p53, we classified cell lines into mutant or wild type, and copy number of SnoN were compared among these two groups. As shown in Figure 6A, we identified significant enrichment of SnoN amplification events in p53 mutant or p53 deleted cell lines (p value: 7.25E-009), indicating a potential association between the two genetic events. To further assess the association of p53 mutation and SnoN amplification, we partitioned cell lines by lineage into 18 sub-groups, and determined the frequency of SnoN amplification and the frequency of p53 mutation within each group and for all groups. A striking correlation between the frequency of p53 mutation and the frequency of SnoN amplification was detected (The Pearson's correlation coefficient was 0.7), where the lineages with higher incidence of p53 mutation tended to have higher incidence of SnoN amplification ( Figure 6B). Thus, at least in human cancer cell lines, increased SnoN expression correlates with inactivation of p53.
Next we asked whether there was any correlation between SnoN expression and the status of p53 in the four types of human cancer tissue samples that showed reduced SnoN expression by staining the cancer tissue arrays with anti-p53. In all normal tissue samples, p53 was absent as expected (data not shown). In contrast, majority of ovarian tumor samples exhibited a strong p53 nuclear staining, indicating the presence of inactivating mutations [24]. The levels of p53 correlated with the malignant stage of the tumors, but showed no significant correlation between SnoN and p53 expression levels (p = 0.817) ( Figure 6C). Similarly, no significant correlation was detected between SnoN expression levels and p53 mutation status in esophageal, breast and pancreatic cancer samples ( Figure 6D). This is not entirely surprising as most cancer tissues we tested seem to downregulate SnoN protein. This downregulation of SnoN protein is sufficient to abolish its tumor suppressor activity and therefore presents no need to additionally inactivate the downstream p53.
Together our study suggests that SnoN is likely to play a tumor suppressor role in the initial stages of human cancer development, and inactivation of this pathway by targeting either SnoN itself or p53 is necessary for malignant progression.

Discussion
In this report, we found that SnoN is expressed in the normal human esophageal, ovarian, pancreatic and breast tissues predominantly in the cytoplasm of both the epithelium and stroma. The intracellular localization of SnoN did not alter significantly in tumor samples, but its expression is downregulated in all four types of tumor epithelia at lower malignant grades, but gradually recovered in higher grade tumors and markedly upregulated in the stroma of esophageal and ovarian cancer tissues. These results suggest that SnoN expression may vary at different stages of tumor progression and that SnoN may play different roles at different stages of malignancy. In particular, the initial downregulation of SnoN in the tumor epithelium is consistent with its antitumorigenic activity. The later upregulation of SnoN in the stroma microenvironment in esophageal and ovarian cancers suggests that it may play a role in promoting tumorigenesis in an indirect manner. Thus, the pro-oncogenic activity of SnoN in human cancer may be non-tumor cell autonomous and through affecting the tumor microenvironment.
Our results differ somewhat from those published previously, in particularly those that reported an increase in SnoN levels in cancer tissues [13,16]. Although the reason for this difference is not known at this time, several possible causes could be speculated. First, the difference could potentially be due to the variations among different samples. Indeed we found that even normal breast tissue samples from different individuals showed a certain level of heterogeneity in term of SnoN levels, possibly due to the different conditions (tissue damage, stress, etc) or treatment procedures that the tissues have experienced. It is therefore quite possible that some cancer samples may also show variations in SnoN levels as a result of the diverse and complex tissue environment under which the tumors develop. However, the overall trend in all four cancer types with over hundreds of tumor samples in our hands is a consistent reduction of SnoN in the tumor epithelium. The level of SnoN may also change depending on the malignant stages of tumors and vary from tissue to tissue. While in esophageal and ovarian cancer, SnoN is downregulated in low grades adenocarcinoma but again elevated in grade III tumors, grade III breast cancer did not show any elevation even when compared to low grade tumors. Similarly, while high SnoN expression is found in stroma of high grade esophageal and ovarian cancer, pancreatic and breast cancer did not show a strong elevation in stroma SnoN expression. Thus, SnoN may play different roles in the progression of different cancer types and possibly through different mechanisms. Another reason may be related to the methods used to measure SnoN expression. Since SnoN expression is upreguated in the stroma while decreased in the tumor epithelium, it is possible that by measuring overall SnoN levels by western blotting, one might detect an increase in the overall expression of SnoN. Finally, the difference could be due to the different antibodies used in the staining. The commercial anti-SnoN antibodies commonly used before showed a high background and sometimes detected nonspecific signals in our hands (data not shown). The anti-SnoN C-terminal peptide antibody has been carefully characterized, and A, 914 cancer cell lines from the Novartis CLE were classified based on their p53 gene status (lost or wild-type) as shown in the X-axis and their correlation with the copy numbers of SnoN as indicated in the Y-axis. A significant enrichment of SnoN amplification events in p53 mutant or deleted cell lines was identified (p = 7.25E-009). B, Cell lines from the CLE were divided into 18 different tissue lineages as depicted by various colors, and the correlation between the frequency of TP53 mutation (X-axis) and frequency of SnoN amplification (Y-axis) was determined to be highly significant with a Pearson's correlation coefficient of 0.7. C, Representative p53 immunohistochemical stain in normal ovarian tissue and ovarian adenocarcinoma of grade I, grade II, and grade III (Original magnification 620). D, Box plot depicting the intensity of epithelial SnoN expression (Yaxis) and p53 protein levels (as marked from 0 to 5, 0 being the lowest level in normal tissues and 5 being the highest). No significant correlation between the status of SnoN protein level and p53 inactivation was noted as measured by the Kruskal-Wallis test (p = 0.817). doi:10.1371/journal.pone.0055794.g006 antigen competition experiments demonstrated the specificity for the SnoN signal ( Figure 1A) [8,12]. Moreover, using this antibody, we have detected dynamic changes in SnoN expression during normal tissue morphogenesis [8,12] and malignant progression, providing additional support that the signals we detect are specific.
Our study has revealed a previously unappreciated role of SnoN in the tumor microenvironment. Especially in esophageal and ovarian tissues, multiple stromal cell types showed high levels of SnoN expression, including smooth muscle cells, blood cells, fibroblasts and infiltrating lymphocytes. Interestingly, the cancers of esophagus and ovary tend to harbor 3q26 amplification and show SnoN copy number increases, and they also display a stronger increase in stroma SnoN levels. Thus, the high SnoN expression in the stroma may be related to gene amplification, and this in turn may enhance malignant progression in these tissues. More and more studies have indicated that tumor-associated stromal cells and infiltrating lymphocytes promote epithelial tumorigenesis, invasion and metastasis, and cancer inflammation [25][26][27]. In addition, infiltrating B-and T-cell lymphocytes can regulate solid tumor development through their abilities to regulate myeloid cell function in solid tumors [28]. Interestingly, TGF-b is a well-known regulator of the tumor microenvironment by modulating the proliferation of fibroblasts, secretion of chemokines and cyokines that control inflammation, recruitment of bone-marrow-derived myeloid cells and host immunosurveillance [29][30][31]. As a negative regulator of TGF-b signaling, it is conceivable that SnoN may also modulate the tumor microenvironment and tumor-host interaction. Our results clearly indicate that this is an important area to focus on in future studies.
High levels of SnoN have been shown to directly stabilize and activate p53. Thus, tissues or cells that show elevated SnoN expression (either due to gene amplification or other means) may cease proliferation due to p53-mediated senescence or cell cycle arrest. Malignant cells have to overcome this barrier in order to maintain their survival and expansion and may do so by either inactivating/deleting SnoN itself or any downstream steps in the SnoN-p53 pathway. Indeed, our analysis of CLE cell lines has indicated a significant correlation between increased SnoN copy number and p53 inactivation, supporting the idea that the tumor suppressive SnoN-p53 pathway must be inactivated to allow malignant progression. A previous study of human breast cancer tissue samples also note a correlation between SnoN upregulation and elevated p53 expression, which is indicative of inativating mutation of p53 [13]. However, in human cancer tissue samples that we have tested, no correlation between SnoN expression and p53 status can be detected. This is not entirely surprising given that these tumor tissues have already downregulated SnoN expression and therefore had no pressing need for additionally inactivating p53.
In summary, we have found that SnoN is expressed in both epithelial and stromal cells in normal human esophageal, ovarian, pancreatic and breast tissues. Unlike what has been depicted in the current models, SnoN expression is downregulated at the protein level in the tumor epithelium in low grade tumors in all four tissues, consistent with its anti-tumorigenic activity. In contrast, SnoN expression is markedly upregulated in tumor stroma in some cancer types, especially at late stages of malignancy. Giving the importance of tumor stroma in influencing malignant progression, this result promopted us to speculate that SnoN may also regulate tumorigenesis through modulating tumor microenvironment.


s2fieldsofstudy: Biology Chemistry

text:
Regulatory Effects of Sestrin 3 (SESN3) in BCR-ABL Expressing Cells

Chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) are characterized by the presence of the BCR-ABL oncoprotein, which leads to activation of a plethora of pro-mitogenic and pro-survival pathways, including the mTOR signaling cascade. We provide evidence that in BCR-ABL expressing cells, treatment with tyrosine kinase inhibitors (TKIs) results in upregulation of mRNA levels and protein expression of sestrin3 (SESN3), a unique cellular inhibitor of mTOR complex 1 (mTORC1). Such upregulation appears to be mediated by regulatory effects on mTOR, as catalytic inhibition of the mTOR kinase also induces SESN3. Catalytic mTOR inhibition also results in upregulation of SESN3 expression in cells harboring the TKI-insensitive T315I-BCR-ABL mutant, which is resistant to imatinib mesylate. Overexpression of SESN3 results in inhibitory effects on different Ph+ leukemic cell lines including KT-1-derived leukemic precursors, indicating that SESN3 mediates anti-leukemic responses in Ph+ cells. Altogether, our findings suggest the existence of a novel mechanism for the generation of antileukemic responses in CML cells, involving upregulation of SESN3 expression.

Introduction
Chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (Ph+ ALL) are characterized by the presence of the BCR-ABL oncoprotein, the product of the Philadelphia chromosome (Ph) resulting from the aberrant translocation between chromosomes 9 and 22 [1,2]. BCR-ABL exhibits constitutively active tyrosine kinase activity, leading to the engagement of many anti-apoptotic and pro-proliferative effector cascades [3,4,5,6,7,8,9]. Specific inhibition of BCR-ABL by first-and second-generation tyrosine kinase inhibitors (TKIs) targeting the kinase domain of BCR-ABL, such as imatinib, nilotinib and dasatinib, has revolutionized the treatment of Ph+ malignancies [10,11]. Despite the potent effects of second-generation BCR-ABL inhibitors, a subset of patients still harbor certain BCR-ABL mutations, such as the T315I mutation, which is refractory to firstand second-generation TKIs in vitro and in vivo [12,13,14]. More extensive identification of patient-specific BCR-ABL mutations and realization of emerging resistance to second-generation TKIs led to rigorous efforts in designing and developing new specific inhibitors that can block the activity of the T315I BCR-ABL mutant. One such agent is the third-generation TKI, ponatinib, as initial studies of ponatinib in CML patients indicated that it has clinical activity with remarkable hematologic and cytogenetic responses even in patients with the BCR-ABL-T315I mutation [15,16]. Despite the success of ponatinib and its recent approval for the treatment of TKI resistant patients, a new study shed light on three newly identified BCR-ABL mutations, L248R, T315V and F317R, detected in CML and Ph+ ALL patients that appear to exhibit differential resistance to TKIs, including the thirdgeneration inhibitors ponatinib and DCC-2036, underscoring the need for novel therapies for resistant Ph+ CML and ALL [17].
While selective targeting of BCR-ABL with new agents may be an effective approach to overcome resistance associated with BCR-ABL mutations, cellular resistance has been documented with mutations unrelated to the BCR-ABL oncoprotein, such as overexpression of the Lyn tyrosine kinase, leading to imatinib resistance by Bcl-2 activation [18,19,20]. A potential strategy to counteract the various modes of resistance could involve targeting signaling cascades that mediate diverse cellular signals, providing an important and possibly more effective approach to reverse leukemic cell resistance in BCR-ABL malignancies.
An important mitogenic pathway engaged by BCR-ABL and readily studied as an alternative therapeutic target is the phosphatidyl inositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway [5,21,22]. mTOR, an evolutionary conserved serine/threonine kinase is the catalytic subunit of two distinct complexes, mTORC1 and mTORC2, with different functions [23,24,25,26,27]. Two recent studies have elucidated the importance of targeting both complexes, as treatment of leukemic cells and patient-derived leukemic progenitors with ATP-competitive inhibitors of mTOR, such as OSI-027 and PP242, elicited strong anti-leukemic responses, even in the case of T315I-BCR-ABL-expressing cells [28,29].
Sestrins are a family of stress-inducible proteins, consisting of Sestrin1 (SESN1), Sestrin2 (SESN2) and Sestrin3 (SESN3). These proteins share high homology with the bacterial AhpD protein, responsible for catalyzing the reduction of peroxiredoxins (Prdx), enzymes that metabolize peroxides [30]. The Sesn1 and Sesn2 genes are under transcriptional control of p53, while SESN3 is transcriptionally regulated by the AKT/FOXO axis, through FOXO1/FOXO3a-mediated gene expression [30,31]. Recently, sestrins have been implicated in the regulation of mTORC1 signaling, as ectopic expression of any of the three sestrins results in inhibition of phosphorylation of mTORC1 pathway effectors [32]. Sestrin-mediated inhibition of mTORC1 occurs via AMPKmediated activation of the TSC1/2 complex [32]. Through an unknown mechanism, sestrins interact with and allow for activation of AMPK, while also facilitating the interaction between AMPK and the TSC1/TSC2 complex [32,33]. Moreover, abolishing either SESN1 or SESN2 expression reverses mTORC1 signaling suppression under basal and stress conditions [32]. The third family member, SESN3, also inhibits mTORC1 as evidenced by the findings that the FOXO1/3a-mediated transcriptional upregulation of SESN3 leads to activation of the AMPK/TSC1/2 axis, thereby inhibiting mTORC1 activity [34]. SESN3 also mediates ROS detoxification by FOXO as well as inhibition of cellular senescence [35].
The initial link between oncogenesis and sestrins arose from the studies of Ras expression, concluding that Ras suppresses the sestrin family of genes [36]. As mTOR signaling is an attractive drug target in a number of malignancies, upon elucidation of the sestrins-mTORC1 crosstalk, studies were conducted focusing on the link between sestrins and cancer via the mTORC1 and AMPK axis. Ablation of drosophila sestrin (dSESN) enhanced TORC1stimulated hyperplastic growth [37], while overexpression of SESN1 or SESN2 suppressed hyperactive mTORC1-mediated malignant cell growth [32,38].
In the present study, we examined the effects of BCR-ABL and mTOR inhibition on SESN3 expression. Our data demonstrate that treatment with first and second-generation TKIs induce expression of SESN3 levels in wild-type BCR-ABL expressing cells, while the third-generation TKI ponatinib induces SESN3 expression even in cells harboring the T315I-BCR-ABL mutant. SESN3 induction is mTOR-dependent, as treatment with the ATP-competitive mTORC1/2 inhibitor OSI-027 also results in SESN3 induction. Importantly, overexpression of SESN3 results in partial inhibition of mTORC1 and exhibits potent suppressive effects on leukemic cells expressing either WT-BCR-ABL or mutant TKI-resistant BCR-ABL, suggesting a mechanism for generation of antileukemic responses.

Cells and Reagents
The human Ph+ ALL cell lines BV173 and BV173R were kindly provided by Dr. Nicholas J. Donato (University of Michigan, Ann Arbor, MI) [28]. The Ba/F3 p210T315I mouse stable transfectants were kindly provided by Dr. Brian J. Druker (Howard Hughes Medical Institute and Oregon Health & Science University, Portland, OR) [28]. The U937 AML cell line was obtained from ATCC. The KT-1, BV173, BV173R, U937 and Ba/F3 cell lines were grown in RPMI 1640 medium supplemented with fetal bovine serum and gentamicin. Antibodies against the phosphorylated forms of AKT, ribosomal protein S6 and ERK and total forms of AKT, ribosomal protein S6 and ERK were purchased from Cell Signaling Technology, Inc. Antibodies against SESN2 and SESN3 were purchased from Proteintech. Antibodies against the phosphorylated form of PRAS40 were purchased from EMD Millipore. Antibodies against the total form of PRAS40 were purchased from Life Technologies. Rapamycin was purchased from Calbiochem/EMD. Imatinib mesylate and ponatinib were purchased from ChemieTek. OSI-027 was purchased from ChemieTek. In some initial studies the compound from OSI-pharmaceuticals was used. Nilotinib was from Chemie Tek. The AKT inhibitor MK-2206 was purchased from Santa Cruz Biotechnology, Inc. (catalog #sc-364537) The SESN2 (catalog #SC128085) and SESN3 (catalog #SC313706) human cDNA clones were obtained from OriGene.

Cell lysis and Immunoblotting
Cell lysis and immunoblotting were performed as in previous studies [28,39]. For such studies, the cells were treated with OSI-027 (5 mM), rapamycin (20 nM), imatinib mesylate (1 or 5 mM as indicated) or nilotinib (100 nM) for the indicated times, unless otherwise specified.

Statistical Analyses
Statistical significance was assessed using paired t-test analysis. p values are indicated in the relevant figures and/or the corresponding figure legends.

Detection of ROS
Detection of ROS was conducted as previously described [41]. Briefly, cells were treated as noted. 30 minutes before end of treatment, half of the cells were collected by centrifugation at 4uC, 400 g for 5 minutes, washed with cold PBS and resuspended in 0.5 mL of cold PBS and kept on ice until the end the treatment (unstained control). The remaining cells were stained with 5 mM DCFDA dye (Invitrogen). At the end of treatment, the remaining cells were collected as described above. ROS measurements were obtained using the BD LSR Fortessa instrument at the RHLCCC Flow Cytometry facility.
Human leukemic hematopoietic progenitor cell assays. Clonogenic hematopoietic progenitor assays in methylcellulose to assess leukemic progenitor colony formation were performed as in previous studies [28,39].
WST-1 assay. Cell proliferation was detected using the cell proliferation reagent WST-1 (Roche) [42]. Briefly, BV173R cells were transiently nucleofected with either empty vector (E.V.) or SESN2 or SESN3 expressing plasmids. 48 hours after transfection, cells were seeded in 96-well plates and proliferation was measured at the time points indicated. First, the cells were treated with WST-1 substrate for 2 hours, then, the cells were analyzed on a microplate reader (BioTek).

Results and Discussion
There have been previous studies implicating AKT in the regulation of SESN3 [34], while other work has implicated the AKT/mTOR pathway as an effector pathway for BCR-ABLmediated responses [6,7,28,29,43,44]. We sought to examine whether BCR-ABL exhibits regulatory effects on the expression of SESN3. Inhibition of BCR-ABL kinase activity using imatinib or nilotinib induced SESN3 expression in the WT-BCR-ABLharboring cell line KT-1 (Fig. 1A). As BCR-ABL-expressing cells were found to have activated mTORC1 and mTORC2 complexes [28,29], with the latter being responsible for AKT activation, we examined the effects of the ATP-competitive, dual mTORC1/2 inhibitor OSI-027 and the selective mTORC1 inhibitor rapamycin on the expression levels of SESN3. Treatment with OSI-027 potently induced SESN3 levels, while rapamycin had minimal effects (Fig. 1B). Consistent with the gene expression data, the BCR-ABL inhibitors imatinib and nilotinib as well as the mTORC1/2 inhibitor OSI-027 increased SESN3 protein levels ( Fig. 1C and 1D respectively), while rapamycin had minimal effects (Fig. 1D). Moreover, imatinib treatment of the non-BCR-ABL expressing AML cell line U937 did not induce SESN3 expression (Fig. 1E), suggesting that the upregulation of SESN3 by imatinib is specific to BCR-ABL expressing cells. Interestingly, TKI treatment did not alter the gene expression of SESN2 (Fig. 1F), while on the contrary mTOR inhibition resulted in some decrease in SESN2 expression (Fig. 1G), indicating differential modulation of expression of different sestrin types in BCR-ABL transformed cells. As SESN3 has been found to be modulated by the AKT/FOXO pathway [34,35], we examined the effects of the specific AKT inhibitor MK-2206, which inhibits AKT kinase activity (Fig. S1A). Treatment of KT-1 cells with the AKT inhibitor MK-2206 induced SESN3 expression, but no effects were observed on SESN2 expression (Fig. S1B).
To further determine whether the kinase activity of BCR-ABL is required for SESN3 expression, we examined the effects of TKIs on SESN3 mRNA expression in cells harboring WT-BCR-ABL as compared to cells harboring the TKI mutant T315I-BCR-ABL.
Effective targeting of both mTORC1 and mTORC2 complexes has been shown to overcome resistance to TKIs and dual mTORC1/2 inhibitors potently targeted cells expressing WT-BCR-BL as well as the T315I mutant of the oncoprotein [28,29]. To determine whether catalytic mTOR inhibition results in SESN3 expression in T315I-BCR-ABL cells, the effects of the dual Figure 2. mTOR inhibition but not BCR-ABL inhibition upregulates SESN3 in T315I-BCR-ABL expressing cells. A. BV173 or BV173R cells were treated with imatinib mesylate (5 mM) for 12 hours. RNA was extracted and expression of SESN3 mRNA was determined by quantitative RT-PCR, using GAPDH for normalization. Data are expressed as fold increase in the treated samples over untreated samples and represent means 6 S.E. of 3 independent experiments. B. BV173 or BV173R cells were treated with either imatinib (5 mM) or nilotinib (100 nM) for 16 hours. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated. C. Ba/F3 cells stably transfected with WT-BCR-ABL or T315I-BCR-ABL were treated with either imatinib (1 mM) or nilotinib (100 nM) for 16 hours. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated. D. BV173 and BV173R cells were treated with OSI-027 (5 mM) for 12 hours. RNA was extracted and expression of SESN3 mRNA was determined by quantitative RT-PCR, using GAPDH for normalization. Data are expressed as fold increase in the treated samples over untreated samples and represent means 6 S.E. of 3 independent experiments. E. BV173 or BV173R cells were treated with OSI-027 (5 mM) for 16 hours. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated. F. Ba/F3 cells stably transfected with WT-BCR-ABL or T315I-BCR-ABL were treated with OSI-027 (5 mM) for 16 hours. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated. doi:10.1371/journal.pone.0078780.g002 SESN3 in Ph+ Leukemia Cells PLOS ONE | www.plosone.org mTORC1/2 inhibitor OSI-027 on SESN3 expression were subsequently examined. OSI-027 treatment induced SESN3 mRNA expression (Fig. 2D) and increased SESN3 protein levels ( Fig. 2E) in both the WT-BCR-ABL-expressing BV173 and in the T315I-BCR-ABL expressing BV173R cells. Similar results were obtained when Ba/F3 stable transfectants for WT-BCR-ABL or T315I-BCR-ABL were used (Fig. 2F).
As the T315I mutation is resistant to first and second-generation TKIs, there has been an emerging focus on the effects of the novel third-generation TKI, ponatinib, which has efficacy against the T315I mutant form of BCR-ABL [15,16]. Ponatinib treatment on T315I-BCR-ABL expressing Ba/F3 cells resulted in induction of SESN3 gene expression in the T315I-BCR-ABL expressing Ba/F3 cells, while imatinib treatment did not (Fig. 3A). Ponatinib also induced SESN3 expression in the Ph+ ALL T315I-BCR-ABLexpressing cell line BV173R (Fig. 3B), underscoring that inhibition of BCR-ABL by this novel third-generation TKI is sufficient for SESN3 induction.
In subsequent experiments we sought to define the effects of sestrin overexpression in BCR-ABL transformed cells. There has previously been evidence for anti-tumorigenic effects of sestrins, primarily linked to modulation of ROS [36], while it is known that BCR-ABL expression results in ROS expression [21]. Thus, we examined the effects of SESN3 expression on ROS production in BCR-ABL transformed cells. Overexpression of SESN3 did not appear to decrease DCFDA staining, indicative of ROS, in this system (Fig. 4A). However, independent of ROS modulation, sestrins have been implicated in mTORC1 signaling regulation [30,33]. We examined whether SESN2 or SESN3 have effects on S6 kinase (S6K) signaling by overexpressing either SESN2 (Fig. 4B) or SESN3 (Fig. 4C). Notably, SESN3 overexpression did not affect mTOR mRNA expression (Fig. 4D). SESN3, but not SESN2 overexpression reduced phosphorylation of rpS6 (Fig. 4E), reflecting inhibition of S6K activity. On the other hand, it did not have significant effects on phosphorylation of AKT on Ser473 (Fig. 4F), indicating that there were no effects on mTORC2 activity. Such effects appear to be specific, as SESN3 did not modulate the MEK/ERK pathway as phosphorylation of ERK was not altered (Fig. 4G). SESN2 overexpression did not induce changes in the phosphorylation of the proteins assessed, indicating that the effects on S6K inhibition are SESN3-specific (Fig. 4).
Sestrins have been shown to have negative effects on mTORC1 signaling [34,35,37,46], whose inhibition has been linked to antileukemic effects in BCR-ABL expressing cells [28,29,39]. To examine whether SESN3 is implicated in the generation of antileukemic responses, we overexpressed SESN3 in KT-1 cells and assessed the effects of such overexpression on leukemic CFU-L precursors, by clonogenic assays in methylcellulose. Overexpression of SESN3 in KT-1 cells (Fig. 5A) resulted in potent suppression of leukemic progenitor (CFU-L) colony formation as compared to controls (Fig. 5B). On the other hand, overexpression of SESN2 did not affect KT-1-derived leukemic progenitor colony formation ( Fig. 5C and 5D), suggesting that SESN3, but not SESN2, is a selective mediator of antileukemic effects on BCR-ABL transformed leukemic precursors. Importantly, SESN3 overexpression (Fig. S2), led to a decrease in proliferation of the T315I-BCR-ABL harboring cell line BV173R (Fig. 5E).
While the sestrin family of proteins was first implicated in redox control [30,31,32], recent studies have been focusing on the role of sestrins in mTORC1 signaling. Particularly, sestrins appear to inhibit mTORC1 in an AMPK-dependent manner [33,37,46,47] and previous studies have implicated SESN1 and SESN2 as negative regulators of mTORC1 activity and neoplastic cell proliferation [32]. However, the third member of the sestrin family of proteins, SESN3, has been studied to a lesser extent. There is some evidence this member of the family may be of importance in systems with high mTORC2 activity, as SESN3 is activated by the FOXO1/3a transcription factors, which are inhibited by the mTORC2/AKT signaling pathway [34,35]. Interestingly, studies using Drosophila SESN (dSESN), showed that hyperactivation of TORC1 signaling induces dSESN [37]. An additional complexity in this mechanism is that TORC1 activation leads to ROS accumulation, resulting in JNK/FOXO signaling activation, which further induces dSESN expression [31,37].
There has been prior evidence that CML and Ph+ ALL cells exhibit increased mTORC1 and mTORC2 activities, as it has been previously shown by us and others [28,29,39], while also harboring increased ROS levels compared to their non-transformed counterparts [21]. This led us to studies to determine whether sestrin expression is modulated by targeting BCR-ABL and/or inhibiting mTOR in Ph+ leukemic cells. To the best of our knowledge, our studies provide the first evidence that BCR-ABL inhibition by several different TKIs (imatinib mesylate, nilotinib, ponatinib) results in induction of SESN3 mRNA and protein expression. Likewise, inhibition of mTORC1/2 by OSI-027 also results in upregulation of SESN3. As the mTORC1/2 pathway is downstream of BCR-ABL [5,48,49], TKI-mediated increase of SESN3 likely results, at least in part, by mTORC1/2 inhibition and may be implicated in the generation of antileukemic responses as evidenced by the inhibitory effects of SESN3 on Ph+ leukemic precursors. Importantly, SESN3 induction by OSI-027 is observed in cells harboring the T315I-BCR-ABL mutant, further supporting our findings and underscoring the potential therapeutic relevance of mTOR targeting in BCR-ABL expressing leukemias, especially in the case of TKI resistance. Further confirmation of the role of BCR-ABL kinase activity in SESN3 suppression stems from studies using the third-generation TKI, ponatinib, which targets the T315I-BCR-ABL mutant, as ponatinib induces SESN3 upregulation in two different cell lines expressing the T315I-BCR-ABL oncoprotein. Future work should precisely define signals downstream of mTOR that control SESN3 expression, and also examine potential antileukemic effects of SESN3 on the 3 newly identified ponatinib-resistant BCR-ABL mutants L248R, T315V and F317R [17]. As our studies demonstrate that SESN3 promotes antileukemic responses, such future efforts may lead to the development of novel antileukemic agents that may selectively promote SESN3 expression and function and could be of value in the treatment of Ph+ leukemias alone or in combination with TKIs and/or mTOR inhibitors.


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Differential Resuscitative Effect of Pyruvate and its Analogues on VBNC (Viable But Non-Culturable) Salmonella

An environmental isolate of Salmonella Enteritidis (SE), grown to the logarithmic phase, rapidly lost culturability by the addition of 3 mM H2O2 to cultures grown in Luria-Bertani (LB) medium; however, some H2O2-treated bacteria regained their culturability in M9 minimal medium, if sodium pyruvate was present at at least 0.3 mM. In addition, most pyruvate analogues, such as bromopyruvate or phenylpyruvate, did not show restoration activity similar to that of pyruvate, except in the case of α-ketobutyrate. Further analysis of the mechanism underlying the resuscitation by pyruvate revealed that although many of the bacteria showed respiratory activity on CTC (5-cyano-2,3-di-(p-tolyl) tetrazolium chloride) reduction with or without pyruvate, the biosynthesis of DNA and protein synthesis were quite different in the presence or absence of pyruvate, i.e., pyruvate endowed the cells with the ability to incorporate much more radio-label into precursors during the resuscitation process. These results suggest that pyruvate is one of the key molecules working in the resuscitation process by taking bacteria from the non-culturable state to the growing and colony-forming state by triggering the synthesis of macromolecules such as DNA and protein.

Salmonella is one of the most frequent causes of foodborne diseases in Japan, and is detected not only in food but also in natural environments, including river water, soil, manure or air-borne dust (4,8,16,19,28). Among Salmonella spp., Salmonella enterica servoar Enteritidis (SE) is the dominant species and has sometimes caused mass food poisoning in Japan (17) and the United States (4), although fatalities due to salmonellosis are low. In consideration of the strong infectivity of Salmonella, a better understanding and appropriate risk management of environmental Salmonella are necessary to control the spread of Salmonella to foods in general. Contamination of Salmonella from the environment to food is an important matter for food safety; however, Salmonella is known to persist and survive in the environment even after exposure to such sanitizers as hydrogen peroxide (H 2 O 2 ) (1), a widely used sanitizing agent.
Since Xu et al. (38) reported their pioneering study concerning the existence of the viable but non-culturable (VBNC) state over 30 years ago, a large number of papers have been published by researchers worldwide, documenting the VBNC phenomenon in a wide variety of bacteria. Many pathogens, such as Escherichia coli, Vibrio cholerae, Vibrio vulnificus, Shigella sonnei, Shigella flexneri, Campylobacter jejuni, Legionella pneumophila (3,5,10,26,27,32,34,36), and Salmonella Enteritidis (9) can enter the VBNC state after exposure to adverse environmental conditions such as high/low temperature, osmotic stress, oxidative stress, and nutritional starvation (2,11,23,26,37). The VBNC state is now generally accepted as a state in which a cell is metabolically active but is incapable of undergoing the cell division necessary to grow and to form a colony on growth media (21,25,29). In this paper, we used the definition of "culturability" as colony-forming activity of the cells on LB-agar plates, and that of "viability" primarily as metabolic activity to reduce 5-cyano-2,3-di-(p-tolyl) tetrazolium chloride (CTC). According to the definition, the VBNC state corresponds to the CTC-reduction-positive but colonyformation-negative state. An assay method based on the resuscitation of VBNC cells has been used to study the factors involved in reversing the VBNC state of several bacterial species. For example, Vibrio vulnicus and Salmonella Typhimurium are resuscitated after a temperature change (14,25), and Enterococcus spp., when grown in tryptic soy broth (TSB; 20). In this report, we show that H 2 O 2 exposure rapidly compelled SE cells to enter the VBNC state. Also, addition of sodium pyruvate to H2O2-treated SE cells effectively resuscitated them from the VBNC state to the growing state.
Sodium pyruvate, a well-known compound and an intermediate key metabolite of glycolysis, is known as a bacterial growth inducer for cells treated with H 2 O 2 (6,13,22,23). Although this up-regulating effect of pyruvate on resuscitation is known, the precise mechanism underlying this pyruvate-mediated promotion of growth of bacteria in the VBNC state is not yet clear.
Previously, we found that stressed Salmonella release a large amount of pyruvate during long-term culture in M9 minimal medium containing a high concentration (0.8%) of glucose, and the cells were later proved to be in the VBNC state (Tanda et al., unpublished data). Consequently, we hypothesized that pyruvate is not only a metabolic intermediate of glycolysis but also a certain repair molecule that functions under various stress conditions (Tanda et al., unpublished data).
In this study, the VBNC state of Salmonella Enteritidis cells was shown to be induced reproducibly by incubating the cells with H2O2. Using these VBNC cells, we examined the resuscitation effect of pyruvate and pyruvate analogues. In addition, the metabolic activities of VBNC cells, such as respiration and macromolecular synthesis (DNA, protein), were examined in the course of the resuscitation process from the VBNC state.

Bacterial cell culture
An environmental isolate of S. Enteritidis (SE) clone, SE Cl#15-1, obtained from CAF Laboratories (Fukuyama, Hiroshima, Japan; 33), was used (33) throughout this study. The bacteria were cultured overnight in 10 mL Luria-Bertani (LB) medium (Difco BD, Sparks, MD, USA) in a 50-mL tube at 37°C, with shaking at 150 strokes min −1 and then suspended in fresh LB medium at OD550 of 0.05. Thereafter, they were incubated at 37°C with shaking at 150 strokes min −1 for 105 min until the growth had reached the mid-logarithmic phase. The precultured bacteria were harvested by centrifugation at 2,850×g for 20 min in a swing rotor-equipped KUBOTA 8920 centrifuge (Kubota, Tokyo, Japan) at 4°C, washed twice with 10 mL ice-cold phosphate-buffered saline without calcium (PBS (−)), pH 7.5, and finally suspended in ice-cold PBS (−) at OD550 of 6.30 (about 1×10 9 cfu mL −1 ).

Oxidative stress exposure
Precultured bacteria were suspended in a 50-mL tube containing 10 mL fresh LB medium to which 0.1-10 mM H2O2 (Wako Pure Chemical Industries, Osaka, Japan) had been added. The cells (1×10 7 cfu mL −1 ) were then incubated at 37°C for 0-60 min, with shaking at 150 strokes min −1 , for exposure to oxidative stress. The bacteria were harvested by centrifugation as described above, and subsequently resuspended in fresh LB medium at 1×10 7 cells mL −1 . After serial dilution with ice-cold PBS (−), 25 μL aliquots were plated on LB-agar plates, which were then incubated at 37°C overnight. The resultant colonies were counted to estimate viable bacterial cell number (cfu), which was used as an indicator of culturability.

Resuscitative effect of pyruvate or pyruvate analogues in liquid culture
The H2O2-treated bacteria were harvested by centrifugation as described above, suspended in M9 medium (Difco) at 1×10 7 cfu mL −1 without or with 0.3-30 mM sodium pyruvate, and then incubated at 37°C for various times up to 60 min. Bacterial growth was monitored both by the density of bacterial solution, measured at 550 nm (OD550) with a spectrophotometer (UV-150-02; Shimadzu, Kyoto, Japan), and by cfu.
Besides pyruvate, pyruvate analogues, such as bromopyruvate, phenylpyruvate, and α-ketobutyrate (Sigma Aldrich, St. Louis, MO, USA) at 30 mM were also evaluated for their resuscitative effects by incubating VBNC cells with each analogue at 37°C for 60 min.
Validation of the respiratory activity of the bacteria after the resuscitation processes using confocal laser-scanning microscopy Bacteria in M9 medium containing 0.4% (w/v) glucose were incubated with the reagents of a Bacstain CTC Rapid Staining Kit (Dojindo, Fukuoka, Japan). CTC is an indicator of bacterial aerobic respiration (7,31). The following procedures were performed essentially in the dark throughout the experiment: the bacteria were left to stand for 60 min in an incubator at 37°C. They were then washed with 500 μL PBS (−) by centrifugation at 18,100×g for 5 min at 4°C in an angle rotor-equipped TOMY MX-160 centrifuge (Tomy Seiko, Tokyo, Japan). Finally, the resultant cell pellets were resuspended in 40 μL PBS (−) and placed in the wells of a sterile, poly-L-lysine-coated 8-well glass slide. The cells were allowed to adhere to the glass by standing the slide on ice for 30 min, and then they were fixed on ice for 30 min with 40 μL of 4% (w/v) paraformaldehyde/PBS (−). Cellular DNA was stained with 5 μL of 20 μg mL −1 DAPI (4',6-diamino-2-phenylindole lactate; Sigma Aldrich), after which the slide was rinsed twice with PBS (−). The samples were embedded in Perma Fluor Mountant Medium (Thermo Scientific, Rockford, IL, USA) and then applied to the slide, which was sealed with a cover glass and thereafter examined under a confocal laser-scanning microscope (LSM510; Carl Zeiss, Oberkochen, Germany).
The viability of the bacterial cell was determined using the following formula:

Validation of the biosynthesis of DNA and protein of SE under resuscitation
Precultured SE cells were suspended in 250 mL LB medium containing 3 mM H2O2 at 1×10 7 cfu mL −1 in a 1,000 mL Erlenmeyer flask and incubated at 37°C with shaking at 150 strokes min −1 , to cause oxidative stress. The bacteria were harvested by centrifugation as described above, and then resuspended in fresh LB medium at 1×10 8 cells mL −1 . Alternatively, the bacteria were resuspended to 1×10 8 cells mL −1 in 0.3 mM sodium pyruvate-containing M9 medium, and then incubated at 37°C for various times up to 60 min. The SE cells undergoing resuscitation were incubated with [methyl-

Statistical analysis
Statistical analysis and estimations of significance of difference between groups with comparable variances were performed by one-way analysis of variance (ANOVA).

Reduction in culturability of SE by H2O2
In this study, we first examined the effect of H 2 O 2 on the culturability of SE cells. As shown in Fig. 1A, incubation of the cells with H2O2 for 60 min had a biphasic and dosedependent effect on viability as assessed in terms of colonyforming units (cfu); Although low concentrations (0.1-1 mM) of H2O2 did not affect the culturability of SE markedly, with 1 mM H 2 O 2 reducing the culturability by only 1/10 of that of the control, H2O2 at higher concentrations, i.e., 3 and 10 mM, significantly reduced it. The cfu value decreased to approximately 1/10 4 of the control with 3 mM H 2 O 2 and became undetectable with 10 mM H2O2.
To determine the optimal exposure time to stress with 3 mM H 2 O 2 , we examined the time-course of incubation up to 60 min. As shown in Fig. 1B, SE cells lost their culturability in an incubation time-dependent manner, with the cfu value decreasing gradually to about 1/10 of the control by 30 min, and then rapidly to 1/2.2×10 3 of the control by 60 min.
Based on these results, exposure to 3 mM H 2 O 2 for 60 min was used as the standard stress condition for SE cells throughout this study.

Restoration of culturability of H2O2-treated SE cells by pyruvate and its analogues
We next examined the ability of various chemicals to resuscitate H2O2-treated bacteria. We first tested whether pyruvate would have any effect on H 2 O 2 -treated cells, because we had earlier found that a large amount of pyruvate accumulated in the culture supernatant of SE cells after their incubation with 0.8% (w/v) glucose in M9 minimal medium, under which condition the cells had lost their culturability almost entirely but retained some metabolic activity in terms of their ability to reduce CTC (Tanda, et al., unpublished data).
As shown in Fig. 2A 2B); however, the optical density (OD550) of the cultures did not increase under these conditions (data not shown). These results suggest that pyruvate increased the number of viable and culturable bacteria due to the increase of the cell population able to divide and form colonies on the LB agar plate after the resuscitation procedures. In addition, even intact bacteria (not treated with H2O2) in the logarithmic phase did not multiply in M9 medium in the presence or absence of pyruvate during 60-min incubation, as monitored by changes in both cfu and OD550 (data not shown).
In addition, pyruvate analogues (bromopyruvate, phenylpyruvate, and α-ketobutyrate) were examined in this system to determine whether they would show the same resuscitative effects as pyruvate. As shown in Fig. 3, α-ketobutyrate increased cfu after incubation, although no significant difference was observed, suggesting the resuscitative effect of α-ketobutyrate other than pyruvate. In its presence, the culturability of H2O2-treated cells was restored, being 7.4fold higher than that of the control. Phenylpyruvate had   no effect, whereas bromopyruvate actually reduced the culturability of H2O2-treated cells to 1/75 of the control, probably through the inhibition of glycolysis by interruption of hexokinase II activity (18). These results suggest that the α-keto carboxy residue seems to be necessary for the resuscitation effect but that the structure of this moiety alone is inadequate to manifest the effect.
We also tested the amount of intracellular peroxide of H 2 O 2 -treated cells to avoid the possibility that pyruvate caused resuscitation by simply degrading intracellular H2O2. Quantitation of H2O2 in cell extracts of SE cells was performed with the PeroXOQuant Quantitative Peroxide Assay Kit (Thermo Scientific). As a result, there was no detectable amount of H2O2 or its adduct by the test with the assay kit, as both H2O2-treated and intact cells and the cell extract of intact SE cells degraded exogenous H2O2 (data not shown). Since α-keto acids, such as pyruvate, degrade H 2 O 2 (12), this resuscitative effect of pyruvate and α-ketobutyrate does not seem to be simply due to its H2O2-degrading effect.

Under these conditions, we investigated various metabolic activities of H2O2-treated cells, including respiratory activity (measured with CTC), DNA synthesis ([ 3 H]-thymidine incorporation) and protein synthesis ([ 35 S]-methionine/[ 35 S]cysteine incorporation), together with culturability (colony formation).
As described in the Introduction, we defined VBNC cells as CTC-reduction-positive but colony-formation-negative cells in this study, and thus estimation of the metabolic activity is essential. The respiratory activity, as indicated by the CTC-reduction products seen by confocal laser-scanning microscopy, was not decreased markedly by treatment with H 2 O 2 . Many cells were positive for CTC reduction products both without (Fig. 4A-4D) and with (Fig. 4E-4H) pyruvate treatment. Based on the results of individual cell counting, H 2 O 2 -treated cells retained their activity at approximately 70% of the control (intact, non-treated; Fig. 5A), suggesting that most of these cells were in the VBNC state. In addition, activity was almost completely (92% of the control) recovered by incubation for resuscitation with or without pyruvate for 60 min, showing that the addition of pyruvate was not required for this recovery. Contrary to the respiration activity monitored by CTC reduction, the addition of 0.3 mM pyruvate to H 2 O 2 -treated cells increased their cfu from 0.02% to 1.0% of the control, whereas the non-pyruvate-treated control remained at 0.02% (Fig. 5B). These results suggest that oxidative stress due to 3 mM H2O2 caused only moderate damage to the respiratory activity of Salmonella, keeping the cells in the VBNC state, and that the addition of pyruvate seemed to enable some cells to form colonies.
On the other hand, unlike in the case of respiratory activity, the synthesis of protein and DNA in H 2 O 2 -treated cells was quite different in the presence and absence of pyruvate during the resuscitation process. When the cells were exposed to oxidative stress due to 3 mM H 2 O 2 , both DNA and protein synthesis decreased to 1.8% and 10.4% of the non-treated control, respectively (Fig. 6A, 6B). The addition of 0.3 mM pyruvate elevated these activities. The increase of DNA synthesis showed biphasic changes, with the synthesis rapidly increasing by 15 min, followed by a gradual increase to 3.2fold higher than the basal level (0 time) by 60 min; however, incubation without pyruvate failed to increase it (Fig. 6A). This rapid increase of DNA synthesis seems to suggest a role for DNA synthesis in the resuscitation process induced by pyruvate.  Protein synthesis also showed a similar but moderate increase during 60 min up to 1.6-fold higher than the basal level (Fig. 6B). The culturability of H 2 O 2 -treated cells in 1,000-mL Erlenmeyer flasks was reduced to 0.0006% of the control (Fig. 6C); however, incubation with 0.3 mM pyruvate time-dependently increased culturability up to 0.11% of the non-treated control in terms of cfu. Compared with the results shown in Fig. 5B, where the culture scale was 1/10 smaller, the SE cells seemed to be exposed to much more severe stress because the cfu of 3 mM H2O2-treated cells were lower under these conditions. However, the concentration of the bacterial culture solution did not seem to affect the resuscitation activity markedly because the extent of resuscitation for 60 min in this large-scale culture (172-fold; Fig. 6C) was somewhat similar to that found for the small-scale culture (52-fold; Fig. 5B). Therefore, assessment of the activities of DNA and protein syntheses in this large-scale culture (Fig.  6A, 6B) seems to have provided comparable information for estimation of the resuscitation process.
Taken together, these results imply that some population of VBNC cells had acquired the ability to return to the culturable state by restoration of the biosynthesis of macromolecules by pyruvate, especially DNA.
In addition, we assayed the incorporation of [3-14 C]pyruvate into H2O2-treated and/or resuscitated SE cells. H2O2treated SE cells incorporated the radioactive pyruvate biphasically, as they had incorporated [ 3 H]-thymidine; pyruvate was rapidly incorporated by 15 min and then gradually by 60 min, similar to the case of [ 3 H]-thymidine (data not shown). In keeping with the result, our study suggests that pyruvate was incorporated by VBNC bacteria, resulting in the restoration of DNA synthesis and cell division.

Discussion
In this paper, we showed that SE cells readily changed from the growing state to the nonculturable state by brief treatment with 3 mM H2O2 during incubation in LB medium, and that these nonculturable cells could be resuscitated with pyruvate. As a result, pyruvate resuscitated the cells in liquid cultures containing M9 medium of at least 0.3 mM ( Fig. 2A-B). To ascertain the chemical properties of pyruvate required for the resuscitation, we next examined pyruvate analogues, such as α-ketobutyrate, bromopyruvate, and phenylpyruvate, determining whether these analogues with α-keto carboxy residues would have a resuscitative effect on H2O2-treated SE cells. Among them, only α-ketobutyrate showed a resuscitative effect similar to that of pyruvate (Fig. 3). This analogue is converted to propionyl-CoA by oxidized ferredoxin and 2-oxobutyrate synthase (EC 1.2.7.2), and subsequently to succinyl-CoA through 3 metabolic processes, and finally integrated into the TCA cycle (15). However, our preliminary experiments suggest that activation of the TCA cycle does not seem to be essentially responsible for resuscitation, because thiamine, a cofactor of the pyruvate dehydrogenase complex that promotes the generation of acetyl-CoA, did not show any significant effect on restoration in the presence of pyruvate (data not shown). In addition, the resuscitative effect of pyruvate did not differ between experiments performed under aerobic and anaerobic conditions where, in the latter, the TCA cycle would be inactive (data not shown); therefore, the generation of energy under aerobic conditions itself does not seem to be linked to the recovery of colony-forming activity. These results seem to suggest a correlation with the reports from other laboratories showing that non-culturable bacteria, induced by low temperature and starvation, are restored to culturability by H2O2-degrading substances such as catalase, pyruvate, and α-ketoglutaric acid (6,(22)(23)(24)30) and that non-culturable bacteria do not use these substances as nutrients, but use chemical materials with a H2O2-degrading effect, such as αketo acid and pyruvate. However, in this study, we showed that bromopyruvate and phenylpyruvate, which possess the α-keto carboxy residue acid structure, did not restore the culturability of H2O2-treated SE cells (Fig. 3). In fact, bromopyruvate strongly decreased the number of cfu from the initial (control) level, probably through interruption of glycolysis as described above. Phenylpyruvate, which is not a metabolic inhibitor, did not change the culturability. In addition, our preliminary experiment showed that these H2O2-treated cells did not retain a detectable amount of H 2 O 2 after H 2 O 2 treatment. These results suggest that the resuscitative effects of α-keto acids and pyruvate are partly related to the cellular metabolism centered on the substrates around glycolysis and/or oxidative phosphorylation, not just degrading intracellular peroxide, as opposed to previous reports (6,(22)(23)(24)30). Supporting this, α-lipoate, N-acetyl-L-cysteine and D-mannitol, which are well-known antioxidant or radical-scavenging reagents, were ineffective in resuscitating H 2 O 2 -treated cells (data not shown). The metabolic activities of the cells were decreased by H2O2 treatment (Figs. 5 and 6); however, there were large differences between culturability and the other biological markers of "viability." Based on the definition of a "viable" state, a large population of H2O2-treated cells was viable, because 70% of the cells showed CTC reduction, and the activities were restored completely to the control levels during incubation without or with 0.3 mM sodium pyruvate due to unknown mechanisms (Fig. 5A). DNA synthesis and cell division, both of which are essential to manifest "culturability," were restored rapidly in 15 min in the former, and gradually but greatly in the latter, only in the presence of 0.3 mM pyruvate (Figs. 5B, 6A, 6C). Protein synthesis was also restored gradually in the presence of pyruvate (Fig.  6B). These results suggest that H2O2 treatement induced a VBNC state in SE cells, and that pyruvate helped to restore DNA synthesis first and then culturability, although we could not completely rule out the possibility that a certain number of bacteria with culturability but with very low such metabolic activity in DNA or protein synthesis would gain the activity during re-incubation with pyruvate. The effect of pyruvate might not have been primarily due to any anti-oxidative effect, but to the triggering of certain metabolic activities necessary for resuscitation. Pyruvate may have activated certain metabolic pathways for energy metabolism, probably those including CTC reduction.
Further studies are required to understand the precise molecular mechanism of the resuscitation of VBNC bacteria by pyruvate and α-ketobutyrte in order to improve the risk management toward pathogenic VBNC cells threatening food safety.