extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Chemistry

text:
Galectin-3 Silencing Inhibits Epirubicin-Induced ATP Binding Cassette Transporters and Activates the Mitochondrial Apoptosis Pathway via β-Catenin/GSK-3β Modulation in Colorectal Carcinoma

Multidrug resistance (MDR), an unfavorable factor compromising the treatment efficacy of anticancer drugs, involves the upregulation of ATP binding cassette (ABC) transporters and induction of galectin-3 signaling. Galectin-3 plays an anti-apoptotic role in many cancer cells and regulates various pathways to activate MDR. Thus, the inhibition of galectin-3 has the potential to enhance the efficacy of the anticancer drug epirubicin. In this study, we examined the effects and mechanisms of silencing galectin-3 via RNA interference (RNAi) on the β-catenin/GSK-3β pathway in human colon adenocarcinoma Caco-2 cells. Galectin-3 knockdown increased the intracellular accumulation of epirubicin in Caco-2 cells; suppressed the mRNA expression of galectin-3, β-catenin, cyclin D1, c-myc, P-glycoprotein (P-gp), MDR-associated protein (MRP) 1, and MRP2; and downregulated the protein expression of P-gp, cyclin D1, galectin-3, β-catenin, c-Myc, and Bcl-2. Moreover, galectin-3 RNAi treatment significantly increased the mRNA level of GSK-3β, Bax, caspase-3, and caspase-9; remarkably increased the Bax-to-Bcl-2 ratio; and upregulated the GSK-3β and Bax protein expressions. Apoptosis was induced by galectin-3 RNAi and/or epirubicin as demonstrated by chromatin condensation, a higher sub-G1 phase proportion, and increased caspase-3 and caspase-9 activity, indicating an intrinsic/mitochondrial apoptosis pathway. Epirubicin-mediated resistance was effectively inhibited via galectin-3 RNAi treatment. However, these phenomena could be rescued after galectin-3 overexpression. We show for the first time that the silencing of galectin-3 sensitizes MDR cells to epirubicin by inhibiting ABC transporters and activating the mitochondrial pathway of apoptosis through modulation of the β-catenin/GSK-3β pathway in human colon cancer cells.

Introduction
Among the various types of cancers, colorectal cancer is a serious and common health problem worldwide. This type of cancer is the third most common visceral malignancy and remains a major cause for cancer deaths due to therapy resistance [1]. The development of colorectal cancer usually occurs through a multistage process involving the mutational activation of oncogenes and inactivation of tumor suppressor genes. More than 90% of tumorigenesis in colorectal cancers begins with mutations in the β-catenin signaling pathway [2].
Multidrug resistance (MDR), an unfavorable factor compromising the treatment efficacy of anticancer drugs, involves the upregulation of ATP binding cassette (ABC) transporters and induction of galectin-3 signaling. To circumvent MDR in cancer cells, several strategies, including P-glycoprotein (P-gp) antagonists, antisense oligonucleotides, ribozymes, and other techniques that modulate MDR-related genes, have been developed [3,4]. However, the long half-life of P-gp (at least 16 h) makes it difficult to achieve complete Pgp dysfunction [5]. To solve this problem, the transfection of MDR cells with siRNAs targeting upstream MDR-related genes in a sequence-specific manner may be promising.
Galectin-3, a β-galactoside-binding protein, exhibits a variety of biological and pathological functions, including effects on RNA processing, cell growth, differentiation, adhesion, apoptosis, immune response, malignant transformation, metastasis, and cancer drug resistance [6]. Galectin-3 is expressed in epithelial and inflammatory cells. However, there are contradictory findings regarding the over-or underexpression of galectin-3 in human colorectal cancer. Galectin-3 overexpression was observed in colorectal carcinoma, corresponding to a positive correlation with cancer progression and metastasis [7][8][9]. Zaia Povegliano et al. have found that a high percentage of galectin-3-stained cells could be observed in the most advanced colon cancer patients and patients with recurrence after surgery and chemotherapy treatment [10]. In contrast, a decrease in galectin-3 expression levels has also been found in colorectal cancer [11,12]. Furthermore, Greco and Iacovazzi et al. have reported that the overexpression of galectin-3 and its ligand 90K in serum could be a useful biomarker for colon cancer transformation [13,14]. Galectin-3 is an important anti-apoptotic effector protein that confers resistance to cancer chemotherapy. The knockdown of galectin-3 expression induced apoptosis in human colorectal cancer cells [9]. In addition, keratinocytes and peritoneal macrophages from galectin-3¯/ ¯ mice are sensitive to apoptotic stimuli relative to those cells from galectin-3 +/+ mice [15]. After leukemia cells were stimulated for apoptosis by cisplatin, galectin-3 expression was upregulated and caused resistance to apoptosis in surviving cells [16]. Moreover, the silencing of galectin-3 with RNAi increased the susceptibility of leukemia cells to apoptosis [16].
Numerous studies focused on the molecular mechanisms of galectin-3 involved in cancer cell chemoresistance [17]. In particular, galectin-3 plays a critical role in the regulation of the expression of cancer-related genes, including cyclin D1 and Akt (also known as Protein Kinase B; PKB) [18]. Galectin-3 upregulates β-catenin expression, increases its nuclear accumulation, and augments Wnt/β-catenin signaling in human colon cancer cells by regulating glycogen synthase kinase-3β (GSK-3β) phosphorylation and activity via the PI3K/Akt pathway [19]. In addition, galectin-3 was identified as a binding partner of β-catenin and T-cell factor 4 (TCF4), which thus activates the Wnt signaling target genes cyclin D1 and c-myc in human breast cancer cells [20]. However, the role of galectin-3 in colon cancer drug resistance is complex and unclear.
In this study, we aimed to decipher the galectin-3 molecular mechanisms involved in colon cancer cell drug resistance. The expression of upstream survival signals (GSK-3β, β-catenin, cmyc and cyclin D1) in human colon adenocarcinoma Caco-2 cells was evaluated after epirubicin treatment with or without galectin-3 knockdown. The changes in downstream efflux transporters, such as P-gp (product of MDR1 gene), MDRassociated protein (MRP) 1, and MRP2, and apoptosisassociated proteins, including Bcl-2, Bax, and caspases, were also analyzed under the same conditions. We aimed to test the possibility of RNAi directed against galectin-3 to suppress pump resistance, induce apoptosis, and enhance the chemosensitivity of Caco-2 cells to epirubicin. This is a pioneer study correlating the knockdown and overexpression of galectin-3 with ABC transporter modulation and apoptosis regulation in colon cancer cells with the MDR spectrum.

Reagents
Short hairpin RNA (shRNA) was purchased from the National RNAi Core Facility, Academia Sinica (Taipei, Taiwan, ROC). The shRNA sequences are listed in Table 1

Cell lines
The Caco-2 cell line was obtained from the Bioresource Collection and Research Center of the Food Industry Research and Development Institute (Hsinchu, Taiwan). Caco-2 cells express multiple efflux transporters, including P-gp, MRP1, MRP2, and Bcl-2 [21]. This colon epithelial adenocarcinoma cell line has been used as a model cancer cell line with MDR phenotype in our lab [22,23]. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS; Hyclone), 0.1 mM nonessential amino acids, and 10,000 units/ml of penicillin/streptomycin (Gibco) at 37 °C in a humidified atmosphere of 5% CO 2 and 95% air.

Construction of galectin-3 shRNA
Galectin-3 shRNA (shGal-3) was constructed using the pLKO.1-puro (Amp + ) vector with an U6 promoter. shGal-3 plasmid DNA was extracted using a plasmid mini kit (Qiagen), and lentiviruses were generated by the RNAi Core of the Research Center of Clinical Medicine, National Cheng Kung University Hospital. Briefly, 293T cells were co-transfected with 5 μg packaging plasmid (pCMVΔR8.91), 0.5 μg envelope plasmid (pMD.G) and 5 μg pLKO.1 shRNA using Lipofectamine 2000 (Invitrogen) for 6 h. After 24 h, the supernatants containing viral particles were harvested and filtered through 0.45 mm filters. The filtered supernatant was mixed with polybrene (Sigma) and added to Caco-2 cells for transduction and subsequent overnight incubation. The transfected cells were incubated with DMEM containing puromycin (Amresco) for three days, and the positive clones were selected after confirmation by galectin-3 western blotting.

Cytotoxicity assay
Growth inhibition of Caco-2 cells was evaluated with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Cells (6×10 3 ) were seeded in 96-well plates. After 48 h of treatment of the control and shGal-3 cells with increasing concentrations of epirubicin (0, 0.01, 0.1, 1, and 10 μg/ml), the cells were incubated with 0.2 mg/ml MTT and maintained in 5% CO 2 in an incubator at 37 °C for an additional 4 h. Dimethyl sulfoxide (100 μl) was added to each well to solubilize the formazan, and the absorbance was measured at 540 nm using a microplate reader (Dynatech, Chantilly, VA, USA). The relative cell viability (%) was calculated by dividing the number of cells incubated with different treatments by the number of control cells. The median inhibitory concentration (IC 50 ), defined as the drug concentration necessary to inhibit cell growth by 50%, was then obtained from the curve. Data were expressed as the means ± standard deviation (S.D.) of six experiments.

Apoptosis detection assay
The Annexin V FITC Apoptosis Detection Kit (Roche, Cambridge, MA) was used. After a 48 h-treatment of the control and shGal-3 cells with or without epirubicin (1 μg/ml), staining was performed with Annexin V-propidium iodide (PI) labeling solution for 15 min at room temperature in the dark. Samples were measured using a flow cytometer (Cell Lab Quanta SC MPL; abbreviated as Quanta SC; Beckman Coulter, Fullerton, CA, USA). Data acquisition and analysis were performed using commercial software (Quanta SC). Early apoptotic cells with intact cell membranes expose phosphatidylserine and are bound to Annexin V-FITC, and these cells exclude PI (Annexin V positive, PI negative). Cells in necrotic or late apoptotic stages are Annexin V-FITC and PI positive.

Observation of chromatin condensation using a fluorescence microscope
Control and shGal-3 cells were plated at 2 × 10 5 cells/well in six-well plates and incubated for 48 h with or without epirubicin (1 μg/ml). The cells were collected and stained with acridine orange (10 mg/ml; Sigma). Samples were then observed using an inverted microscope (Eclipse TS-100; Nikon Co., Tokyo, Japan) equipped with a fluorescence image capture device (C-SHG; Nikon) controlled with the Image-Pro Plus software (Media Cybernetics, Inc., Bethesda, MD, USA). Fragmented nuclei and condensed chromatin characteristics were examined and compared with those of the control.

Cell cycle analysis
Control and shGal-3 cells at 10 5 cells/well were incubated for 48 h with or without epirubicin (1 μg/ml). The cells were harvested by centrifugation and gently fixed with 70% ethanol at -20 °C overnight. After fixation, the cells were stained with propidium iodide (1 mg/ml) and incubated for 30 min at 25 °C in the dark. The cells were analyzed using a flow cytometer (Quanta SC). The presented results are from three individual experiments.

Intracellular epirubicin accumulation
The functional involvement of MDR transporters in the efflux of epirubicin was evaluated using a flow cytometer. Control and shGal-3 cells were seeded in six-well plates at 2×10 5 cells/well and allowed to attach overnight. After pretreatment with epirubicin for 48 h, the fluorescent epirubicin that accumulated in Caco-2 cells was measured using a flow cytometer (Quanta SC). Data acquisition and analysis were performed using commercial software (Quanta SC), and determinations were performed in quadruplicate.

Construction and overexpression of galectin-3
The 741-bp human galectin-3 (hGal-3) full-length sequence was amplified by PCR with the following primers: 5´-GAGCGGCCGATGGCAGACAATTTTTCGCTC CA-3( forward) and 5´-GAGGGATCCTTATATCATGGTATATGAAGCAC-3´ (reverse). The gel-purified PCR product was digested using Bam HI and Xho I and then cloned into the pIRES-hrGFP-2a vector (Stratagene Corp., La Jolla, CA, USA) using T4 DNA ligase (Promega, Madison, WI, USA). All of the resulting plasmids were amplified in E. coli DH5α. Successful construction of the pIRES-hGal-3 vector was confirmed using DNA electrophoresis and direct sequencing, which showed a similarity of 99.9%. Caco-2 cells were plated at 1 × 10 5 cells/well in six-well plates and allowed to attach overnight. We mixed 2 μg/well of the pIRES-hGal-3 gene constructs with 6 μl/well of the PolyJet DNA transfection reagent (SignaGen Laboratories, Ijamsville, MD, USA) in 200 μl DMEM. Caco-2 cells were then transfected with the mixture at 25 °C for 30 min and incubated at 37 °C for 48 h.

Real-time PCR of P-gp, MRP1, MRP2, Bax, Bcl-2, and caspases
Control and shGal-3 cells were treated with or without epirubicin (1 μg/ml) for 48 h. Total RNA was extracted from cells using the Total RNA Extraction Miniprep System (Viogene, Taipei, Taiwan) according to the manufacturer's instructions. The RNA yield and purity were assessed using a NanoDrop 2000 (Thermo, Wilmington, DE, USA). cDNA was prepared from total RNA using the High-capacity RNA-to-cDNA kit (Applied Biosystems; Foster City, CA, USA) following the manufacturer's protocol. The gene-specific primers (Table 1) for galectin-3, GSK-3ß, ß-catenin, Cyclin D1, c-myc, MDR1, MRP1, MRP2, Bcl-2, Bax, caspase-3, caspase-8, and caspase-9 were constructed by multiple sequence alignment. 18s rRNA was served as an internal control. Quantitative PCR was conducted using the StepOne Real-Time PCR system (Applied Biosystems) and SYBR Green PCR Master Mix (Applied Biosystems). The cycling program was performed as follows: denaturation at 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The specificity of the gene-specific PCR primers was verified by melting curve and agarose gel analyses. The results presented are from three individual experiments in which each sample was assayed in triplicate and normalized to the 18s rRNA level. Gene expression was calculated as the mRNA expression ratio compared with control cells.

Statistical analysis
Data are presented as the means ± standard deviation. Student's t-test was used to analyze differences between two treatment groups. Statistical analysis was conducted using one-way ANOVA and Dunnett's multiple comparison tests. Significant difference was set at P < 0.05.

Combined epirubicin and shGal-3 treatment significantly intensified epirubicin cytotoxicity
First, Caco-2 cells were treated with epirubicin for 24 h, and the expression of galectin-3 and P-gp was analyzed by western blotting. As shown in Figures 1A and 1B, epirubicin significantly increased the expression of galectin-3 and P-gp. We then established a galectin-3 stable knockdown cell line by transfecting Caco-2 cells with shGal-3 for further studies. After 48 h treatment of the control and shGal-3 cells with increasing concentrations of epirubicin (0, 0.01, 0.1, 1, and 10 μg/ml), the relative cell viability (%) of the Caco-2 cells was evaluated with an MTT assay ( Figure 1C). The epirubicin potency for cellgrowth inhibition was expressed as the IC 50 value. The mean IC 50 value for epirubicin treatment was 0.91 ± 0.03 μg/ml. In addition, the IC 50 value for the combined epirubicin and shGal-3 treatment was 0.07 ± 0.01 μg/ml, which is remarkably lower than that for epirubicin treatment alone. This combination enhanced epirubicin cytotoxicity and allowed for a reduced epirubicin dose.

Combined epirubicin and shGal-3 treatment induced apoptosis-mediated morphological changes in Caco-2 cells and increased the sub-G 1 accumulation of Caco-2 cell cycle
Flow cytometric analysis using annexin V/PI staining showed that cells treated with epirubicin or shGal-3 alone exhibited a significant increase in apoptosis compared with the control (Figure 2A). The epirubicin and shGal-3 combination treatment remarkably promoted apoptosis with an increased amount of Roles of Galectin-3 in Multiple Drug Resistance PLOS ONE | www.plosone.org positive staining for membrane asymmetry and PI uptake (Figure 2A).
Caco-2 cells were treated with shGal-3 and/or 1 μg/ml epirubicin for 48 h and observed by fluorescence microscopy. The viable control cells and cells treated with scrambled shRNA had consistently bright green nuclei ( Figure 2B).
Caco-2 cells exposed to shGal-3 and/or epirubicin displayed plasma membrane blebbing, cell shrinkage, and concentrated green areas of condensed or fragmented chromatin in the nucleus ( Figure 2B). The appearance of apoptotic bodies after shGal-3 and/or epirubicin treatment along with the apoptosis detection assay further verified that the cytotoxic effects of Caco-2 cells treated with shGal-3 and/or epirubicin exhibited a typical cell cycle phase distribution when examined with a flow cytometer. The proportion of cells exhibiting sub-G 1 fluorescence, which corresponds to the apoptotic cell subpopulation, significantly increased after treatment with shGal-3 and/or epirubicin for 48 h ( Figure 2C). The percentage of the sub-G1 phase cells after combined treatment (51.3 ± 4.2%; P < 0.05) was significantly higher than that for cells treated with epirubicin (12.1 ± 1.4%) or shGal-3 (32.2 ± 1.1%). Thus, the silencing of galectin-3 by RNAi improved the ability of epirubicin-induced apoptosis in Caco-2 cells.

shGal-3 significantly increased the intracellular epirubicin retention in Caco-2 cells
As pump-resistance efflux transporters, P-gp, MRP1, and MRP2 actively transport MDR substrates such as epirubicin out of cells and decrease the intracellular accumulation of epirubicin in cancer cells [3,22,24]. These phenomena mediate the development of pump-related MDR [25,26]. We found that the inhibition of pump resistance by shGal-3 treatment resulted in high epirubicin accumulation in Caco-2 cells as determined by flow cytometry (Figure 3A; P < 0.05). However, scrambled shRNA had no obvious effect on epirubicin retention in Caco-2 cells ( Figure 3B).  The mean fluorescence intensity of Epi was normalized to 100%. The mean fluorescence intensity of Epi plus shGal-3 was normalized relative to Epi. *, P < 0.05 compared with control and #, P < 0.05 compared with Epi. The data are presented as the means ± SD from three independent experiments.

galectin-3 and β-catenin but increased the expression of GSK-3β
Cells treated with shGal-3 inhibited the mRNA expression level of galectin-3 and β-catenin but increased the expression of GSK-3β ( Figure 4A). The same trend could also be observed for the combined treatment (i.e., shGal-3 and epirubicin). In contrast, the transfection of a galectin-3 overexpression vector into Caco-2 cells decreased the GSK-3β mRNA level but slightly increased the mRNA level of galectin-3 and β-catenin. We also found that epirubicin treatment increased the mRNA expression of galectin-3 and GSK-3β but decreased the expression of β-catenin. These results indicated that the expression of galectin-3 and β-catenin mRNA in Caco-2 cells was upregulated and downregulated, respectively, with epirubicin treatment. In addition, the knockdown of galectin-3 upregulated the GSK-3β mRNA level, which was accompanied by the downregulation of β-catenin.

Combined shGal-3 and epirubicin treatment inhibited the mRNA expression level of the cyclin D1 and c-myc induced by the epirubicin treatment
We further investigated the expression of the Wnt signalingrelated genes cyclin D1 and c-myc upon galectin-3 knockdown and/or epirubicin treatment. The knockdown of galectin-3 slightly decreased the mRNA expression level of cyclin D1 and c-myc (P < 0.05; Figure 4B), whereas epirubicin significantly upregulated their expression (P < 0.05; Figure 4B). However, the combined treatment effectively reduced the mRNA expression of cyclin D1 and c-myc, which had been remarkably induced by epirubicin treatment. Moreover, the mRNA expression level of cyclin D1 induced by the combined treatment was higher than that of the control (P < 0.05; Figure  4B), but it was significantly lower than that for epirubicin treatment alone (P < 0.05). These results imply that cyclin D1mediated epirubicin resistance is partially reversed by combined treatment (Figure 4B). The mRNA expression level of c-myc almost returned to the level of the control after combined treatment. Finally, these phenomena could be rescued after transfection with a galectin-3 overexpression vector.

Combined shGal-3 and epirubicin treatment significantly inhibited the mRNA expression level of MDR1, MRP1, and MRP2
Next, we tried to understand whether the mRNA expression of the epirubicin-induced pump resistance proteins MDR1, MRP1, and MRP2 were regulated by galectin-3. We found that the corresponding mRNA expression level of MDR1, MRP1, and MRP2 was significantly increased by epirubicin treatment (P < 0.05), whereas that of MDR1 and MRP1 (P < 0.05) was moderately reduced after galectin-3 knockdown ( Figure 4C). The reduction in the MDR1, MRP1, and MRP2 mRNA expression level was also observed for the combined treatment ( Figure 4C, P < 0.05) and could be reversed after galectin-3 overexpression. These findings indicate that epirubicin-induced mRNA expression of MDR1, MRP1, and MRP2 is regulated by galectin-3 ( Figure 4C).

Combined shGal-3 and epirubicin treatment changed the mRNA expression level of Bax, Bcl-2, and caspases
In addition, we examined the expression of Bax, Bcl-2 and caspases upon shGal-3 and/or epirubicin treatment. The knockdown of galectin-3 increased Bax and reduced Bcl-2 mRNA expression, which leads to apoptosis ( Figure 5A; P < 0.05). In contrast, the overexpression of galectin-3 reversed these phenomena. Treatment with epirubicin alone significantly modulated the corresponding mRNA level of Bax, caspase-3, and caspase-9 ( Figure 5A and 6; P < 0.05). In addition, the knockdown of galectin-3 remarkably increased the Bax-to-Bcl-2 ratio ( Figure 5B; P < 0.05). However, due to the development of resistance, epirubicin reduced the expression of Bax and increased the level of Bcl-2. Furthermore, the knockdown of galectin-3 partially reversed epirubicin-mediated resistance.

Confirm the protein expression level of the Wnt/βcatenin signaling pathway and apoptosis-associated pathway components after epirubicin treatment in wild type or galectin-3 knockdown Caco-2 cells
To verify the results observed in real-time PCR, we further examined the expression level of Wnt signaling pathway and apoptosis-associated proteins by western blotting (Figure 7). In accordance with previous findings, GSK-3β and Bax were upregulated after the treatment of galectin-3 knockdown cells with epirubicin for 48 h. The simultaneous downregulation of pGSK-3β, β-catenin, c Myc, and the pro-apoptotic protein Bcl-2 was also found. In addition, the expression of the anti-apoptotic protein Bcl-2 was significantly reduced in galectin-3 knockdown cells. It should be noted that the upregulation of GSK-3β and Bax and the down regulation of pGSK-3β, β-catenin, c Myc, and Bcl-2 could also be found at 2, 4, 6, 12, and 24 h after epirubicin treatment (Figure 7).

The knockdown of galectin-3 in Caco-2 cells inhibited the P-g and cyclin-D1 protein expression after epirubicin treatment
Because activation of the β-catenin signaling pathway by GSK-3β inhibition can increase the expression of P-gp, we knocked down or overexpressed the upstream regulator galectin-3 to decrease the GSK-3β activity by transfecting Caco-2 cells with galectin-3 siRNA or a galectin-3 expression vector. We found that the P-gp expression was concomitantly increased with the elevation of cyclin D1 in galectin-3 overexpressing cells. The same results could also be observed in epirubicin-treated cells. In addition, The knockdown of galectin-3 could reduce the expression of cyclin D1 and the downstream product P-gp ( Figure 8).

Combined shGal-3 and epirubicin treatment increased the caspase-3 and caspase-9 activity
Finally, we evaluated the activity of the caspase-3, caspase-8, and caspase-9 induced in galectin-3 knockdown cells with or without epirubicin treatment to determine the apoptotic pathway involved. The activity of caspase-3 and caspase-9 was enhanced by shGal-3 or epirubicin alone treatment (P < 0.05) and further increased by the combined treatment ( Figure 9; P < 0.05). However, the activity of caspase-8 was not influenced by galectin-3 knockdown, epirubicin, or combined treatment.  [27,28]. Overexpressed Pgp and MRPs such as MRP1 and MRP2, members of ABC transporters, are the main causes of pump-related MDR. TCF4/ β-catenin responsive elements are found in the promoter of the human MDR1 gene, corresponding to a positive correlation between the expression of β-catenin, c-myc, and cyclin D1 and the upregulation of P-gp [29][30][31]. c-Myc can activate MDR1 transcription by binding to the E-box motif located in the MDR1 gene promoter, thus positively controlling MDR expression [32]. Knockdown of c-myc significantly downregulates c-myc and Pgp expression and results in an increase in doxorubicin uptake [33].The expression of MDR1 can also be upregulated by glucosylceramide synthase (GCS) through the β-catenin signaling pathway [34], and a reduction in the GCS activity by inhibitors can induce GSK-3-regulated apoptosis via the accumulation of endogenous ceramide [35]. Phosphorylation of GSK-3β at serine 9 inactivates GSK-3β activity and causes the stabilization and activation of β-catenin signaling, which subsequently enhances P-gp expression [31,36]. Furthermore, the stable transfection of a cyclin D1 antisense construct into human pancreatic cancer cells leads to decreased levels of the mRNA of chemoresistance genes such as MDR1 and MRP [37].
The upregulation of the antiapoptotic survival defensive system is one of the primary non-pump related MDR mechanisms. Although the anti-apoptotic role and mechanism of galectin-3 in cancer drug resistance have been studied [38], the regulatory pathway and mechanism(s) of chemoresistanceassociated proteins modulated by galectin-3 in MDR cancer cells have not been fully elucidated. Previous reports have shown that galectin-3 mediates nuclear β-catenin accumulation and subsequently increases TCF4 transcriptional activity followed by the upregulation of its target genes, such as cyclin D1 and c-myc, in colon cancer cells [9,19]. Yamamoto-Sugitani et al. also showed that high level of galectin-3 expression in chronic myelogenous leukemia prompted drug resistance through activation of Akt and Erk [39]. Knockdown of galectin-3 inhibits phosphorylation of GSK-3β and increases GSK-3β expression, which leads to β-catenin phosphorylation and results in the degradation of β-catenin by the proteasome [19,20,40]. In addition, GSK-3β is also involved in cyclin D1 mRNA transcription and ubiquitin-dependent proteolysis [41].
In this study, we found that Caco-2 cells reduced epirubicininduced apoptosis not only through an increase in galectin-3 expression but also through the activation of P-gp ( Figure 1). The reduced expression of galectin-3 significantly enhanced the cytotoxicity of the epirubicin treatment ( Figure 1) and increased apoptosis (Figure 2). Western blotting and real-time PCR results showed that galectin-3 silencing resulted in decreased levels of phospho-GSK-3β at serine 9 and increased GSK-3β and GSK-3α expression. The increased expression of GSK-3β led to the downregulation of β-catenin and, subsequently, cyclin D1 and c-myc. These findings indicated that the knockdown of galectin-3 inhibits the expression of Wnt downstream effectors including β-catenin, cyclin D1 and c-myc at the mRNA and protein levels. We also found that the combined treatment of epirubicin and shGal-3 efficiently reversed the MDR transporter-mediated resistance induced by epirubicin administration as demonstrated by epirubicin accumulation, real-time PCR, western blotting, and MTT assays. These data suggest that shGal-3 downregulates the expression of cyclin D1 and c-myc, which play important roles as oncogenes and MDR/MRPs transcriptional upregulators. In accordance with these observations, the silencing of galectin-3 effectively inhibits the expression of MDR1, MRP1, and MRP2, thus circumventing pump-resistance and enhancing the sensitivity of Caco-2 cells to chemotherapy. This is the first demonstration showing that the silencing of galectin-3 may inhibit MDR through the simultaneous suppression of ABC transporters, such as P-gp, MRP1, and MRP2 via the β-catenin/GSK-3β pathway.
In addition to understanding the role of galectin-3 in cancer drug resistance, we further examined the key role of shGal-3 in apoptosis induction. Cheong et al. have reported that silencing of galectin-3 augmented apoptosis induction with chemotherapy by decreasing the expression of cell survival molecules such as survivin and cyclin D1 in gastric cancer cells [42]. Suppression of cyclin D1 has also been demonstrated to inhibit tumor cell growth in human hepatocellular carcinoma and colonic adenocarcinoma [43]. Our findings demonstrated that shGal-3 and/or epirubicin treatment triggered the apoptosis of Caco-2 cells. The combined treatment of epirubicin and shGal-3 exhibited a better apoptosis-inducing effect than that of epirubicin and shGal-3 treatment alone. Galectin-3 silencing interrupted the β-catenin pathway and inhibited cyclin D1 and c-myc, which play critical roles in cell cycle regulation.
Consistent with these models, our results support the idea that the suppression of cyclin D1 and c-myc inhibits the growth of Caco-2 cells and increases their chemosensitivity to epirubicin.
The caspase family of cysteine proteases subsequently mediated an apoptosis cascade corresponding to changes in the cell cycle and morphological observations as determined with a flow cytometer and fluorescence microscope. Caspase-9 is an initiator caspase for the mitochondrial pathway whereas caspase-8 is an initiator caspase for the death receptor pathway, and both of these pathways share downstream effector caspases such as caspase-3 and caspase -7 [3,23]. According to our findings, shGal-3 and/or epirubicin treatment triggered apoptosis through the intrinsic mitochondrial pathway (a negligible change in caspase-8), which is in agreement with previous studies [3,22,23]. The proposed scheme of molecular mechanisms involved in galectin-3 silencing is shown in Figure  10.
In conclusion, we show for the first time that galectin-3 silencing sensitizes MDR cells to epirubicin by inhibiting P-gp and MRPs and the activation of the mitochondrial apoptosis pathway through modulation of the β-catenin/GSK-3β pathway in human colon cancer cells. The complicated regulation of MDR highlights the need for a multifunctional approach for the


extfieldsofstudy: Biology Medicine

text:
Germline Genetic Variation Modulates Tumor Progression and Metastasis in a Mouse Model of Neuroendocrine Prostate Carcinoma

Neuroendocrine (NE) differentiation has gained increased attention as a prostate cancer (PC) prognostic marker. The aim of this study is to determine whether host germline genetic variation influences tumor progression and metastasis in C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of aggressive NEPC. TRAMP mice were crossed to the eight progenitor strains of the Collaborative Cross recombinant inbred panel to address this. Tumor growth and metastasis burden were quantified in heterozygous transgene positive F1 male mice at 30 weeks of age. Compared to wild-type C57BL/6J-Tg(TRAMP)824Ng/J males, TRAMP x CAST/EiJ, TRAMP x NOD/ShiLtJ and TRAMP x NZO/HlLtJ F1 males displayed significant increases in tumor growth. Conversely, TRAMP x WSB/EiJ and TRAMP x PWK/PhJ F1 males displayed significant reductions in tumor growth. Interestingly, despite reduced tumor burden, TRAMP x WSB/EiJ males had an increased nodal metastasis burden. Patterns of distant pulmonary metastasis tended to follow the same patterns as that of local dissemination in each of the strains. All tumors and metastases displayed positive staining for NE markers, synaptophysin, and FOXA2. These experiments conclusively demonstrate that the introduction of germline variation by breeding modulates tumor growth, local metastasis burden, and distant metastasis frequency in this model of NEPC. These strains will be useful as model systems to facilitate the identification of germline modifier genes that promote the development of aggressive forms of PC.

Introduction
Prostate cancer (PC) is the second leading cause of cancer mortality in men [1]. Despite the reduced mortality that has arisen with an increased availability of tools for early diagnosis and the success of hormone ablation therapies, advanced and recurrent PC remains incurable. Conventional assessment of PC prognosis relies heavily upon the Gleason grading scale to assess the histological severity of the primary tumor. However, this pathological grading system suffers from significant interobserver variability, and as a consequence, there is a need for additional means of assessing prognosis.
Neuroendocrine differentiation (NED), which is defined as the emergence of single or small clusters of neuroendocrine cells in conventional prostatic adenocarcinomas through trans-differentiation, has been gaining increasing attention as a potential prognostic marker [2]. Adenocarcinomas with significant NED are typically associated with a poorer prognosis, with NED being increased in high-grade and high stage tumors, and particularly in hormone treated and castration resistant disease [3]. Neuroendocrine prostate carcinomas (NEPC), which includes carcinoid tumor and small-cell carcinoma, are heterogeneous tumors containing mainly NE cells, and are associated with a poor outcome [2,4]. Although NEPC are rare and comprise less than 2% of all tumors [5,6], a sizable subset of conventional prostate adenocarcinomas displays NED. One plausible factor influencing the induction of NED is germline genetic variation. Despite the fact that recent studies have identified novel genes orchestrating this aggressive disease [2,7,8], it remains elusive that how host germline genetic variation influences susceptibility to NED or NEPC.
The influence of germline variation on tumor progression has been proven in other cancer types by introducing germline polymorphism into mouse models of cancer by breeding. This type of strategy was utilized in the FVB/N-Tg(MMTV-PyVT)634Mul/ J mouse model of mammary tumorigenesis to identify progression and metastasis quantitative trait loci (QTLs). Subsequent fine mapping studies allowed for the characterization of Brd4, Sipa1 and Rrp1b as novel breast cancer metastasis susceptibility genes [9,10,11,12]. The clinical significance of these was established by demonstrating that germline polymorphisms within these genes are associated with poor prognosis in multiple human breast cancer cohorts [13,14,15].
The TRAMP transgenic mouse model was utilized in this study to investigate the role of host germline polymorphism in the development of aggressive prostate carcinoma. This model utilizes the androgen-responsive minimal probasin promoter (PB) to initiate SV40 virus T antigen (TAg)-mediated tumorigenesis in the prostatic epithelium. It is a particularly useful model for studying progression and metastasis, since by 30 weeks of age mice typically display a high incidence of multi-organ metastases, which is not a characteristic of any other prostate cancer mouse model [16,17]. Also, studies have shown that NE phenotype tumors in TRAMP mice share certain key molecular features with NED and NEPC in humans, such as tumor growth regulation by high expression of AURKA, MYCN, FOXA2 and SIAH2 [7,8,18].
In this study, germline polymorphism was introduced by breeding to determine the extent to which host genetic variation influences tumor progression and metastasis in this model. Specifically, TRAMP mice were crossed to the eight progenitor strains of the Collaborative Cross (CC) recombinant inbred panel, which are a genetically diverse mapping panel that captures nearly 90% of the known variation present in laboratory mice [19,20] This study conclusively demonstrates that the introduction of germline variation by breeding significantly modulates tumor growth, local metastasis burden, and distant metastasis frequency in TRAMP mice.

Animal Husbandry and Genotyping
C57BL/6J-Tg(TRAMP)824Ng/J (TRAMP-B6) mice and eight CC progenitor strains, C57BL/6J (B6), A/J (A), 129S1/SvImJ (129S), NOD/LtJ (NOD), NZO/HlLtJ (NZO), CAST/EiJ (CAST), PWK/PhJ (PWK) and WSB/EiJ (WSB), were obtained from Jackson Laboratories (Bar Harbor, ME). TRAMP-B6 females were crossed with eight CC progenitor strains to generate eight different F1 strains of mice (TRAMP-B6, TRAMP-A,  TRAMP-129S, TRAMP-NOD, TRAMP-NZO, TRAMP-CAST, TRAMP-PWK and TRAMP-WSB, respectively) hemizygous for PB-TAg transgene. TRAMP-B6 females were bred with nontransgenic B6 males for colony maintenance. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the National Human Genome Research Institute Animal Care and Use Committee (protocol number: G-09-2). All necropsies were performed under sodium pentobarbital anesthesia followed by cervical dislocation, and all efforts were made to minimize suffering. The mouse tail genomic DNA was extracted from F1 progeny with HotSHOT method [21] for genotyping analysis. To identify the hemizygous PB-TAg transgene positive F1 (TRAMP F1) mice, PCR screening was performed as described [22].

Tissue Collection
TRAMP F1 male mice were sacrificed by pentobarbital overdose at 30 weeks of age or humane endpoint, whichever were achieved first. Humane experimental endpoints for this study were rapid weight loss, hunched posture, labored breathing, trauma, impaired mobility, dysuria, or difficulty in obtaining food or water. Prostatic tumor, seminal vesicles, lungs, liver, and lymph nodes were harvested from TRAMP F1 males. Prostatic tumor and seminal vesicles were weighed to quantify tumor burden. Tumor and seminal vesicle weights were normalized to age of the mouse at the time of euthanasia by multiplying the actual weight by the inverse ratio of the age in days at the time of death and the pre-designated experimental endpoint (i.e. 210 days). Visible enlarged lymph nodes in para-aortic region were weighed to quantify metastatic lymph node burden. These were again normalized to age of the mouse at the time of euthanasia. Lungs were collected to determine isolated tumor cell infiltrates in lung parenchyma and microscopic metastatic lesions. Other organs displaying macroscopic metastatic lesions through gross observation were also collected for histology. These collected tissues were fixed in buffered formalin (10% w/v phosphate buffered formaldehyde, Fisher Scientific) or Z-fix (zinc buffered formaldehyde, Anatech Ltd.) overnight and then transferred to 70% ethanol. Fixed tissues were embedded in paraffin, sectioned to a thickness of 4 mm and stained with hematoxylin and eosin (H&E). Histology slides were scanned with Scanscope Digital microscope (Aperio, Vista, CA).

Immunohistochemistry
Tissues were collected and sectioned as described above for immunohistochemical (IHC) analyses. Histopathological analyses of these tissues were undertaken by Dr. A. Molinolo. Tissues were stained with the following primary antibodies: anti-synaptophysin

Time Course Experiments
TRAMP F1 males of B6, WSB, PWK and NOD backgrounds were allocated to the groups and separated throughout different time points. For each of these strains of TRAMP F1 mice, three mice were assigned for each time point. These mice were sacrificed at 4, 8, 12, 16, 20 and 25 weeks; prostate and seminal vesicles were harvested for histopathological analysis. Prostatic intraepithelial neoplasia (PIN) lesion in TRAMP mice has been termed as atypical hyperplasia of TAg in previous literature [23]. However, lesions of prostate epithelium were categorized into three categories, hyperplasia, PIN and carcinoma, following the conventional classification [4]. Prostates from these F1 males were collected at 8 weeks of age and subjected to IHC staining with anti-TAg to determine the strain specific differences in TAg expression in prostatic epithelium.

Quantitative and Statistical Analysis
MedCalc (Mariakerke, Belgium) was used to analyze phenotype data. Statistical significance levels for observed differences were calculated with the Mann-Whitney U test. Kaplan-Meyer survival curves were plotted for survival analyses and hazard ratios calculated using the Cox proportional hazards model. Imagescope software (Aperio, Vista, CA) was used to quantify the nuclear expression of TAg in prostatic epithelium. The significance levels for the strain specific differences in TAg expression were determined with Fisher's exact test. Correlation coefficient and level of its significance were equated with Spearman's rank analysis. P,0.05 was considered significant.

Germline Genetic Variation Modulates Tumor Associated Mortality and Tumor Burden in TRAMP Mice
The aim was to assess tumor growth and metastasis in TRAMP F1 males at 30 weeks of age. However this goal was frequently not achievable owing to factors relating to the overall health of the mouse necessitating euthanasia for humane reasons listed above. Tumor-associated mortality was therefore followed for TRAMP F1 mice, and significant differences were observed in a number of the F1 strains (Fig. 1a). Compared to wildtype C57BL/6J-Tg(TRAMP)824Ng/J males, tumor associated mortality was significantly higher in TRAMP-NOD (hazard ratio [ TRAMP-A, TRAMP-129S, TRAMP-WSB and TRAMP-PWK males did not show any significant difference in their survival times in comparison to wildtype C57BL/6J-Tg(TRAMP)824Ng/J males.
To account for differences in tumor associated mortality, tumor weights were adjusted in all strains to account for age of the mouse at the time of euthanasia/death. Compared to wildtype C57BL/ 6J-Tg(TRAMP)824Ng/J males (age adjusted mean 6 SEM of tumor burden = 1.5460.65 g, n = 35), both TRAMP-NOD (n = 42) and TRAMP-NZO (n = 57) males displayed a significant increase in tumor growth (tumor burden = 9.8661.16 g and 5.6660.97 g, respectively; P,0.001; Fig. 1b). TRAMP-CAST animals (n = 24) also displayed a significant increase in tumor growth, albeit at a somewhat lesser level of significance (tumor burden = 4.5361.09 g; P = 0.050; Fig. 1b). Conversely, both TRAMP-WSB (n = 68) and TRAMP-PWK (n = 37) males displayed a significant reduction in tumor growth (tumor burden = 0.7960.31 g [P = 0.001] and 0.0260.01 g [P,0.001], respectively; Fig. 1b). Indeed, only 29/68 TRAMP-WSB mice and 1/37 TRAMP-PWK mice developed macroscopic tumors, weighing $0.2 g. TRAMP-129S (n = 35) and TRAMP-A (n = 68) did not show significant difference in tumor growth compared to wildtype TRAMP mice. These data conclusively demonstrate that the introduction of germline variation by breeding significantly modulated tumor burden in the TRAMP mouse.

Germline Variation does not Affect TAg Expression in Prostatic Epithelium
The dramatic reduction in prostate tumor burden seen in the TRAMP-PWK and TRAMP-WSB strains compared to other F1 mice could be due to suppression of TAg transgene in the prostatic epithelium. To eliminate this possibility, IHC analysis for TAg transgene expression was performed using prostates from 8 week old wildtype C57BL/6J-Tg(TRAMP)824Ng/J, TRAMP-PWK, TRAMP-WSB and TRAMP-NOD mice. The percentage of prostatic acinar cells expressing TAg from anterior, lateral, ventral and dorsal lobes was quantified. The total amount of cells expressing TAg in the prostate did not vary significantly in TRAMP-PWK, TRAMP-WSB and TRAMP-NOD mice compared to wildtype C57BL/6J-Tg(TRAMP)824Ng/J (P.0.1; Fig.  S1). These data indicate that TAg expression does not differ among strains and that the observed phenotypic differences are due to germline genetic variation.

Strain Specific Differences in Prostate Carcinoma Metastasis
Prostate carcinoma in the TRAMP model exhibits multi-organ metastasis, primarily to lymph nodes and also to lungs, liver, kidney and bones [16]. In order to examine the effect of strainspecific modifier loci on tumor cell capacity to disseminate to distant organs via lymphatic and blood vessels, the incidence of metastasis were tracked in pelvic and renal lymph nodes, lungs, liver and kidney in TRAMP F1 mice. Strain-specific variation was observed in the incidence of metastases in multiple organs (Fig. 2a  and 2b). IHC for SV40-TAg and androgen receptor were performed to better characterize these lesions. Regardless of strain background, all metastases stained positive for SV40-TAg and negative for AR (Fig. S2).
A high incidence of metastasis was observed in some F1 strains. Several of the F1 strains exhibited striking differences in age adjusted local nodal metastasis burden (Fig. 2c) compared to the actual frequency of nodal metastasis (Fig. 2a). The wildtype C57BL/6J-Tg(TRAMP)824Ng/J strain exhibited the lowest age adjusted local metastasis burden with an average local nodal weight of 0.1260.02 g. Five F1 strains had a significantly increased age adjusted local nodal metastasis compared to this: TRAMP-NZO (met burden = 0.

Characterization of the NE Phenotype in TRAMP Tumors
NE phenotype prostate tumor cells in TRAMP mice display similar morphological and molecular characteristics to human NED and NEPC cells, including the expression of IHC markers, synaptophysin, chromogranin A and FOXA2, and loss of expression of cytokeratin 8 (CK8) [2,4,7,8,26]. IHC for the NE marker synaptophysin was performed in tumors and metastatic lesions. Regardless of strain background, all macroscopic tumors ($0.2 g) stained positive for synaptophysin (Fig. 3). Tumor cells infiltrating lung, liver and lymph node tissues also stained positive for synaptophysin (Fig. 4). These observations establish that tumor burden and nodal metastatic burden, used as quantitative traits in this study, represent phenotypes of NEPC.
To further characterize the NE phenotype, IHC was performed for the transcription factors FOXA1 and FOXA2, and the luminal epithelial marker CK8. As expected, CK8 staining was absent in all TRAMP F1 tumors (data not shown). Staining for FOXA2 mirrored that of synaptophysin, and was observed in all tumors. However, such consistency was not observed when comparing FOXA1 and FOXA2 staining patterns with some foci staining strongly for both FOXA1 and FOXA2, while others stained strongly for only either one of these transcription markers (Fig. 3 gl). Such differences in FOXA1 and FOXA2 staining were not strain-dependent and most likely represent heterogeneity of the overall process of tumorigenesis.

Germline Factors Mediate NE Carcinoma Initiation
To investigate differences in tumor initiation and progression, serial histological analysis of prostate and seminal vesicles was performed in some of the strains with the most divergent phenotypes compared to the wildtype C57BL/6J-Tg(TRAMP)824Ng/J strain (TRAMP-NOD, TRAMP-WSB and TRAMP-PWK). Hyperplastic lesions were evident in dorsolateral and anterior prostates in all strains as early as 4 weeks. By 8 weeks of age, these hyperplastic lesions invariably progressed to PIN. The severity and number of hyperplastic foci and PIN lesions increased with age in these F1 mice, and no strain differences were noted.
Seminal vesicle hyperplasia was benign in nature and progressed to become papillary adenomas or phyllodes-like tumor consisting of stromal and epithelial proliferation protruding into the glandular lumen. NE carcinomas consist of solid masses of tumor cells with atypical and pleomorphic cells without nuclear polarity and scant cytoplasm. The histological features of NE carcinoma and seminal vesicle hyperplasia did not differ between wildtype C57BL/6J-Tg(TRAMP)824Ng/J, TRAMP-NOD, TRAMP-WSB and TRAMP-PWK strains. Similarly, histological features did not differ from wildtype C57BL/6J-Tg(TRAMP)824Ng/J mice in all other F1 strains studied (data not shown). NE carcinomas were found to be arising from ventral and/or dorsolateral lobes of prostate in TRAMP mice. These strains exhibited differences in the incidence of seminal vesicle hyperplasia and NE carcinomas (Table S1). The earliest time point when the NE lesion was evident in TRAMP-NOD mice was 8 weeks. The incidence of NE carcinoma increased with age, and by 16 weeks, 100% of TRAMP-NOD mice (6/6) displayed NE carcinoma lesions or tumor. Conversely, only 1 out of 15 wildtype C57BL/6J-Tg(TRAMP)824Ng/J males developed NE tumor at 16 weeks of age. In our time-course study, all TRAMP-WSB mice developed seminal vesicle hyperplasia by 25 weeks of age (6/6), but none had NE carcinoma. Given that no tumors were evident in TRAMP-PWK mice by 30 weeks of age, a set of these mice were aged to 50 weeks (n = 4). Interestingly, only 1 of these TRAMP-PWK mice developed NE tumors and died at 38 weeks of age. Another TRAMP-PWK mouse developed only hyperplasia, and 2 of these 4 TRAMP-PWK developed severe hyperplasia and phyllodes-like proliferations at 50 weeks. These data emphasize previous observations that NE lesions arise independently of the PIN lesions in the TRAMP model [23]. More importantly, the observed strain-specific differences demonstrate that paternal genotypes substantially affect the initiation and kinetics of NE tumorigenesis in TRAMP F1 mice.

Discussion
Although NE carcinoma represents only 1-2% of prostate malignancies, this form of PC is highly aggressive and has a poor survival rate [3,27]. Additionally, NED is present in almost .90% of high grade adenocarcinomas of prostate [28]. More importantly, androgen depletion therapy strongly stimulates NED [29,30]. The characteristics of these types of tumor, including aggressive tumor growth, multi-organ metastases and relapse of disease after castration or androgen depletion are well recapitulated by TRAMP mouse model [24,31,32]. The overall aim of this work is to identify germline susceptibility genes that are associated with an increased risk of NED development. The potential clinical significance of this is that it may prove possible to type polymorphic germline modifier genes at the time of PC diagnosis to more accurately identify men at risk of progression, and thus initiate more aggressive therapies in these metastasis-prone individuals.
Germline predisposition to diseases displaying a complex, non-Mendelian inheritance pattern is controlled by polymorphisms within multiple genes, each of which likely have a small effect in isolation [33]. The identification of modifier genes of tumor progression and metastasis has proven particularly difficult using genome wide and familial association studies. An alternate approach has been to map disease modifier loci using mouse models of human disease [34,35]. Selection of the appropriate mouse strain(s) for this type of study is of critical importance to maximize genetic power and increase the likelihood of identifying genes relevant to human disease [36,37]. This study was carried out to determine whether germline modifiers modulate the development of aggressive NEPC in TRAMP model. Our data have proven that this is indeed the case, and the information gained from these analyses will be used to direct future genetic analyses.
In this study, polymorphisms carried by NOD males were shown to increase tumor growth and dissemination. Conversely, progression was significantly suppressed by modifiers inherited from PWK males, with NE tumorigenesis being almost completely suppressed in this strain. A particularly interesting observation was that TRAMP-CAST and TRAMP-WSB strains displayed a particularly high propensity for metastasis, where the nodal metastasis burden frequently exceeded the primary tumor weight (Fig. 2C). Indeed, the disease course in the TRAMP-WSB strain was particularly unusual when one considers that only 29/68 developed macroscopic tumorigenesis and that the overall age adjusted prostate and seminal vesicle tumor burden in this strain was significantly lower than wildtype C57BL/6J-Tg(TRAMP)824Ng/J mice (see Fig. 1a and 1b, respectively). The dissociation of tumor growth and metastasis in this strain underscores the differing mechanisms involved in these two processes and makes TRAMP-WSB a particularly important strain for future genetic mapping studies.
Additionally, our time-course experiment data showed that prostate and seminal vesicle tumor initiation are controlled by inherited polymorphic genes in these strains. This time-course study demonstrated that there is dissociation among the experimental groups between the development of neuroendocrine prostate tumors, which are high in the TRAMP-NOD strain but low in the other groups and seminal vesicle hyperplasia, which is high in the wildtype C57BL/6J-Tg(TRAMP)824Ng/J and TRAMP-WSB strains but low in TRAMP-NOD and TRAMP-PWK. Overall, these data imply that these differing components of tumorigenesis in TRAMP mice are influenced by different germline polymorphisms. Interestingly, another study reported that NE carcinoma occurs in 20% of TRAMP mice on B6 background and 100% of mice on FVB background, and in ,50% of TRAMP-B6 x FVB F1 mice. This suggests that the interaction of modifiers present in B6 mice decreased NEPC incidence compared to TRAMP-FVB mice [23]. However, the present study demonstrates that modifiers present in NOD mice can override the suppressive effect of B6 strain modifiers to promote NEPC incidence. Collectively, the presented data demonstrate that polymorphic genes derived from paternal strains likely influence tumor progression at multiple stages of disease development.
We do however acknowledge that the TRAMP model suffers a number of limitations. Specifically, the histological features of TRAMP tumors are much more reminiscent of human NEPC, which is relatively rare in humans, rather than the far more common adenocarcinoma with NED. The precise origins of the neoplastic process in the TRAMP mouse were clearly described in the seminal work of Chiaverotti et. al. [23]. This study determined that two distinct neoplastic cellular lineages evolve within TRAMP tumors. The first lineage, which was termed 'abnormal hyperplasia of TAg', was determined to follow a benign course and does not progress beyond pre-malignant PIN lesions. The second lineage, which accounts for the aggressive malignancies observed in TRAMP mice, arises spontaneously and has a strong neuroendocrine phenotype. Our work confirms the findings of this important study. Specifically, although abnormal hyperplasia of TAg and PIN was observed in all F1 animals, the incidence or kinetics of these did not differ between strains. As observed by Chiaverotti et. al. [23], strain differences appear to exert the majority of their influence upon the NE component of TRAMP tumorigenesis. Another limitation of this model is the relative infrequency of skeletal metastases [16]. Initial analyses demonstrated a slight increase in the frequency of metastases to long bones in more F1 mice displaying a more aggressive phenotype (e.g. TRAMP-NOD). However, the overall incidence of bone metastases remained low and it is unlikely that formal quantification would yield any data of value in the cohort sizes described here.
Regardless of the limitations of the TRAMP model, we argue that the modifier genes identified in future studies performed on the basis of the experiments performed here are likely to be applicable not only to NEPC but also more generally to the pathogenesis of aggressive human PC. Prostate adenocarcinomas and NE carcinomas derived from both human and mice all possess the capability to metastasize to lymph nodes, lungs and liver. This indicates that these two distinct tumor cell types may well have acquired similar molecular characteristics under the influence of common allelic determinants to promote a more metastatic phenotype. This is supported by the fact that metastatic disease is enhanced in recurrent PC after androgen depletion therapy, which is similar to the highly metastatic behavior displayed by NEPC cells lacking androgen receptors [38]. In addition, both androgen-independent PC and NEPC cells over-express transcription factors such as FOXA1, indicating the involvement of common factors in the transcriptional regulation in these cells [39,40,41]. These observations support the notion that common pathways may well be involved in the modulation of adenocarcinoma-associated NED and NE prostate cancer progression.
In conclusion, this study establishes that germline polymorphisms are significant modulators of NE tumor initiation and growth kinetics in the TRAMP model. TAg-mediated tumorigenesis and metastasis have been characterized in the progenitor strains of the CC recombinant inbred panel, which will facilitate future modifier mapping studies. Identification of the candidate genes and characterization of genetic polymorphisms associated with this aggressive form of prostate disease will facilitate an improved understanding of this aspect of PC pathogenesis and potentially facilitate the identification of individuals at increased risk of NED. Additionally, selective use of these TRAMP F1 strains in preclinical chemotherapeutic trials for specifically targeting metastasis or tumor growth of NEPC may prove to be a valuable tool.

Supporting Information
Figure S1 Strain specific differences in the expression of TAg transgene in prostate epithelia of TRAMP F1 mice. Transgene was found to be expressing predominantly in ventral and dorsolateral prostates (representative photomicrographs (a), (b) and (c)) of TRAMP F1 mice. (c). Percentage of cells showing positive IHC staining with anti-TAg across different strains of F1 mice from ventral, dorsal, lateral and anterior lobes of prostate collected from 8 weeks old TRAMP F1 mice. Bar graph represents average percent of cells stained positive 6 SD for TRAMP F1 strains. Bar denotes 50 mm.
(TIF) Figure S2 Representative IHC analysis of TRAMP F1 metastatic lesions. IHC analysis for androgen receptor (AR) and SV40-TAg was performed to better characterize metastatic lesions in TRAMP F1 strains. With regards to AR, immunostaining was positive in a high proportion of the nuclei in a paraffin embedded section of normal prostate (a). No staining was seen in either lymph nodes (c) or lung metastases (e), regardless of strain background. For the SV40-TAg, the normal prostate control is not immunoreactive (b), but strong positivity was seen in the nuclei of both lymph nodes (d) and lung metastases (f). The arrows point to the metastatic area embedded in the lung parenchyma. Bar denotes 50 mm. (TIF)


s2fieldsofstudy: Biology Medicine Chemistry

text:
Blockade of VEGFR-1 and VEGFR-2 Enhances Paclitaxel Sensitivity in Gastric Cancer Cells

Purpose Hypoxia-inducible factor-1α (HIF-1α) increases transcription of the vascular endothelial growth factor (VEGF) gene. Inhibition of VEGF abolishes VEGF mediated induction of HIF-1α. Recent reports suggested that HIF-1α also mediated the induction of class III β-tubulin (TUBB3) in hypoxia. TUBB3 confers resistance to taxanes. Inhibition of VEGF may decrease the expression of HIF-1α and TUBB3. This study was undertaken to investigate the roles of vascular endothelial growth factor receptor (VEGFR) in gastric cancer cell behavior and to identify methods to overcome paclitaxel resistance in vitro. Materials and Methods The protein expression levels of HIF-1α and TUBB3 were measured in human gastric cancer cell lines (AGS) under normoxic and hypoxic conditions. The relationship between TUBB3 and paclitaxel resistance was assessed with small interfering TUBB3 RNA. AGS cells were treated with anti-VEGFR-1, anti-VEGFR-2, placental growth factor (PlGF), bevacizuamb, and paclitaxel. Results Hypoxia induced paclitaxel resistance was decreased by knockdown of TUBB3. Induction of HIF-1α and TUBB3 in AGS is VEGFR-1 mediated and PlGF dependent. Hypoxia-dependent upregulation of HIF-1α and TUBB3 was reduced in response to paclitaxel treatment. Expressions of HIF-1α and TUBB3 were most decreased when AGS cells were treated with a combination of paclitaxel and anti-VEGFR-1. AGS cell cytotoxicity was most increased in response to paclitaxel, anti-VEGFR-1, and anti-VEGFR-2. Conclusion We suggest that blockade of VEGFR-1 and VEGFR-2 enhances paclitaxel sensitivity in TUBB3-expressing gastric cancer cells.

INTRODUCTION
Although the incidence and mortality of gastric cancer have decreased in the Western world, gastric cancer persists as a common malignancy and leading cause of cancer-related death in Asian contries. 1,2 Surgery is the only curative treatment for gastric cancer. Chemotherapy achieves favorable results for unresectable advanced vival and overall response rate, but not associated with significant increases in overall survival. 25 To date, we had insufficient evidences for the benefit of adding bevacizumab to chemotherapy in the treatment of gastric cancer.
Placental growth factor (PlGF) was discovered shortly after VEGF. Whereas VEGF binds both VEGFR-1 and VEGFR-2, PlGF binds to VEGFR-1 but not VEGFR-2. The key role of VEGF and its receptor VEGFR-2 in tumor angiogenesis is firmly established, but the contribution of VEGFR-1 remains poorly defined. 26 In the present study, we investigated the relationships between VEGF, HIF-1α, and TUBB3 in gastric cancer cells (AGS). We report that blockade of VEGFR-1 and VEGFR-2 with concomitant paclitaxel treatment increase the cell cytotoxicity of TUBB3expressing gastric cancer cells.

MATERIALS AND METHODS Drugs and reagents
Paclitaxel (BMS Pharmaceuticals, New York, NY, USA) and bevacizumab (Roche Pharmaceuticals, Basel, Switzerland) were diluted in absolute DMSO. This solution was diluted to 0.1% DMSO on each day of experimentation. The PlGF and VEGFR-1/2 neutralizing monoclonal antibodies were purchased from research and development systems.

Cell culture and hypoxic treatment
The human gastric cancer cell lines, AGS, SNU, and TMK1 were used as in vitro model systems. Cells were cultured in RPMI 1640 with 10% fetal bovine serum. For normoxia, cultures were incubated at 37°C in a humidified chamber of 5% CO 2 . For hypoxia, cells were treated with CoCl 2 . Confirmatory experiments under hypoxic conditions (1% O 2 /5% CO 2 /94% N 2 ) were performed using a Modular Incubator Chamber-101. Each hypoxia experiment was conducted under CoCl 2 -treated and true hypoxic conditions, and the results were equivalent.
Taxanes such as paclitaxel and docetaxel are a class of anticancer agents that bind to the β tubulin subunit of polymerized microtubules and induce hyperstabilization, which causes cell cycle arrest and apoptosis. 3 Taxanes are used most commonly for the treatment of breast, lung, ovary, and gastric cancer. The taxanes paclitaxel and docetaxel exhibit similar efficacies in gastric cancer treatment. [4][5][6][7][8][9][10][11] There are at least eight mammalian β tubulin isotypes, and the isotype composition in humans varies across tissues. 12 Overexpression of class III β tubulin (TUBB3) has been observed in several human cancer cell lines such as prostate, ovarian, breast, and non small cell lung cancer. Cells expressing TUBB3 show resistance to docetaxel and paclitaxel. [12][13][14][15] A small cohort study of advanced gastric cancer patients who were receiving preoperative docetaxelbased chemotherapy detected a correlation between expression of TUBB3 and poor response to chemotherapy. 16 Hypoxia in solid tumors is associated with resistance to chemotherapy, induction of angiogenesis, and poor patient prognosis, and angiogenesis is a hallmark of human malignancies. The induction of vascular endothelial growth factor (VEGF), mediated by interacting genetic and environmental signals, is an essential component of tumor angiogenesis. 17 The transcription factor hypoxia-inducible factor-1α (HIF-1α) is a primary regulator of VEGF during hypoxic conditions, and Calvani, et al. 18 reported that inhibition of VEGF or the VEGF receptor-2 (VEGFR-2), abolishes VEGF-mediated induction of HIF-1α. That is, HIF-1α increases VEGF induction, and VEGF also induces HIF-1α expression. A recent report suggested that HIF-1α also mediates TUBB3 induction in hypoxia. [19][20][21] Through these results, we postulated that blockade of VEGF in gastric cancer cells would be associated with a decrease in HIF-1α and TUBB3 expression and a concomitant increase in sensitivity to paclitaxel.
The response of gastric cancer cells to anti-VEGF antibodies is of interest because bevacizumab, a humanized monoclonal antibody against VEGF, is used currently as an anti-angiogenic treatment for metastatic colorectal cancer, non-squamous non-small cell lung cancer, and metastatic breast cancer. [22][23][24] Recent the Avastin in Gastric Cancer trial evaluated the efficacy of adding bevacizumab to chemotherapy in the first-line treatment of advanced gastric cancer. Adding bevacizumab to chemotherapy was associated with significant increases in progression free sur-containing assembled tubulin. Each fraction was separated by SDS-PAGE. Immunoblots then were performed using anti-pan-α-tubulin rabbit polyclonal antibody (Santa Cruz Biotechonology, Santa Cruz, CA, USA).

Statistical analysis
The Student's t-test was used to detect significant differences among treatment groups. Statistical significance was assigned at p<0.05.

Induction of HIF-1α, TUBB3 and VEGF in hypoxia
The expression levels of TUBB3 and VEGF were measured during the induction of HIF-1α in hypoxic AGS. AGS exhibited well-defined hypoxic induction of HIF-1α, VEGF, and TUBB3 compared with other cell lines (SNU638, TMK1) ( Fig. 1).

TUBB3 confers gastric cancer cells with resistance to paclitaxel
To assess whether TUBB3 expressions is associated with chemotherapeutic drug resistance in AGS, TUBB3 was knocked down with small interfering RNA (siTUBB3), and the effects of paclitaxel on AGS growth under normoxic and hypoxic conditions were evaluated over a 24-48 h period. AGS was strongly resistant to paclitaxel during hypoxia. Knockdown of TUBB3 decreased the resistance to paclitaxel during hypoxia, and cytotoxicity was increased (Fig. 2).

VEGFR-1 signals increase HIF-1α and TUBB3 expressions in AGS cells
After treatment with paclitaxel, cytoskeletal component were isolated from AGS cells, and tubulin polymerization was monitored. Western blots were performed using a pan α-tubulin polyclonal antibody to probe lysates from cells that had been treated with paclitaxel or paclitaxel and beva-

Cell proliferation assay
To estimate viability, 10 5 cells were seeded in 24-well plates, subjected to normoxia or hypoxia, and exposed to neutralizing antibodies in the culture media for 4 h. Cells then were treated with bevacizuamb and/or paclitaxel for 24 h. Cells were incubated for 24, 48, or 72 h, and EZ-CyTox (Daeil Lab Service, Seoul, Korea) was added to each well. After 3 h, absorbances at 420 nm were measured using a microplate reader (EL800, BioTek, Winooski, VT, USA).

Tubulin polymerization assay
Cells were treated with various concentrations of drugs for 24 h, washed twice with phosphate buffered saline, and lysed for 5 min in 100 μL hypotonic buffer [1 mM MgCl 2 , 2 mM EGTA, 0.5% NP40, 20 mM Tris-HCL (pH 6.8), protease and phosphatase inhibitors]. Samples then were centrifuged at 16000×g for 10 min at room temperature. Supernatants, containing soluble tubulin, were separated from pellets, Fig. 1. Induction of HIF-1α and its target genes, VEGF and TUBB3, in human gastric cancer cell lines under hypoxia. HIF-1α, VEGF and TUBB3 expression levels were increased during hypoxia, compared to normoxia. Actin was used as a loading control. Band intensities were quantified by densitometry. HIF-1α, hypoxia inducible factor-1α; VEGF, vascular endothelial growth factor; TUBB3, class III β-tubulin. cizumab under normoxic conditions or after 24 h of hypoxia. Paclitaxel activity was inhibited in hypoxia. Tubulin polymerization was decreased in hypoxia compared with normoxia and it was statistically significant (p<0.01). Hypoxic decrease of polymerization was lessened after treated with bevacizumab (Fig. 3A).
As previously mentioned, we postulated that blockade of VEGF in AGS would be associated with a decrease in HIF-1α and TUBB3 expression and a concomitant increase in sensitivity to paclitaxel. We treated cells with bevacizumab, anti-VEGFR-1 and anti-VEGFR-2 neutralizing antibodies to test this hypothesis. AGS cells were cultured under normoxic and hypoxic conditions for 24 h without exogenous growth factors, in the absence or presence of anti-VEGFR-1, anti-VEGFR-2, or bevacizumab with simultaneous paclitaxel treatment. HIF-1α and TUBB3 were increased in a hypoxic condition. Paclitaxel treatment decreased the hypoxia-dependent increased expression of HIF-1α and TUBB3. When paclitaxel was combined with bevacizumab or anti-VEGFR-2, expression levels of HIF-1α and TUBB3 were further decreased. The greatest decrease in HIF-1α and TUBB3 levels occurred in response to paclitaxel and anti-VEGFR-1 treatment in AGS cells (Fig. 3B). Our results using AGS cells  Fig. 3. VEGFR-1/2 contributes to tubulin polymerization. (A) Western blots using a pan α-tubulin polyclonal antibody to probe cell lysates treated with paclitaxel with or without bevacizumab for 24 h during normoxia or hypoxia. Paclitaxel activity was inhibited in hypoxia. Tubulin polymerization was decreased in hypoxia compared with normoxia and it was statistically significant. Cells were treated with paclitaxel with/without bevacizumab at 100 nM and 100 ug/mL, respectively. Lane 1: no drug, 2: paclitaxel, 3: paclitaxel with bevacizumab. Actin was used as a loading control. Band intensities were quantified by densitometry. (B) Hypoxia-dependent upregulation of HIF-1α and TUBB3 was most reversed in the presence of paclitaxel and anti-VEGFR-1 for 24 h. HIF-1α was measured from nuclear fractions, and TUBB3 was measured from total cell lysates. Band intensities were quantified by densitometry. *p<0.01. HIF-1α, hypoxia inducible factor-1α; TUBB3, class III β-tubulin; VEGFR, vascular endothelial growth factor receptor. without anti-VEGFR-1, anti-VEGFR-2, and bevacizumab with simultaneous paclitaxel exposure. The cell viability assay demonstrated that AGS cell cytotoxicity was increased in response to combined treatment with paclitaxel and bevacizumab compared with paclitaxel treatment alone. Cytotoxicity was increased synergistically in response to simultaneous treatment with paclitaxel, anti-VEGFR-1, and anti-VEGFR-2 (p<0.05) (Fig. 5).

DISCUSSION
Bevacizumab is accepted as the standard treatment for advanced metastatic colorectal cancer, but to date is not considered as the standard treatment of gastric cancer. 24 VEGF can bind with the receptor tyrosine kinases, VEGFR-1 and VEGFR-2. The key roles of VEGF and its receptor VEG-FR-2 in tumor angiogenesis have been established. 26,27 Activation of VEGFR-2 leads to auto-phosphorylation and the activation of downstream signaling pathways, such as Raf/ MEK/ERK and PI3K/Akt kinase cascades. 25 In contrast, the function of VEGFR-1 remains poorly defined. VEG-FR-1 has been associated with tumor growth, tumor cell ac-contrast with results using HCT116 cells, in which hypoxiainduced HIF-1α was inhibited via VEGFR-2. 18 To examine VEGFR-1 function, we investigated whether the VEGFR-1 ligand, PlGF, influences the expression of HIF-1α in AGS and HCT116 cells. Cells were cultured under normoxic or hypoxic conditions in the presence of PlGF (range, 0.1-2.0 ng/mL). HIF-1α expression was increased in hypoxia and was dose dependently more increased according to the concentration of PlGF (Fig. 4A). In contrast with AGS cells, PlGF rarely influenced HIF-1α expression in HCT116 cells. AGS cells were cultured under normoxic and hypoxic conditions in the absence or presence of PlGF and anti-VEGFR-1. Expression levels of HIF-1α and TUBB3 were increased in hypoxia and were more increased in response to PlGF treatment. The increased expressions of HIF-1α and TUBB3 were suppressed when anti-VEGFR-1 antibodies were combined with PlGF treatment (Fig. 4B). We concluded that induction of HIF-1α in AGS is VEGFR-1 mediated and PlGF dependent. HIF-1α is measured from nuclear fractions, and TUBB3 is measured from total cell lysates. The observed increases in HIF-1α and TUBB3 expression levels were decreased when anti-VEGFR-1 was combined with PlGF treatment. Band intensities were quantified by densitometry. *p<0.05, † p<0.01. PlGF, placental growth factor; HIF-1α, hypoxia inducible factor-1α; TUBB3, class III β-tubulin; VEGFR, vascular endothelial growth factor receptor.

A B
sults, we suggest that AGS cells express functional VEGFR-1 and VEGFR-2. TUBB3 confers chemoresistance to taxanes. Our experiment results of paclitaxel resistance in AGS cells are consistent with other reports suggesting that TUBB3 affects chemoresistance to taxanes in diverse cancer cells. [12][13][14][15][16] Raspaglio, et al. 21 also reported that TUBB3 is not only a parameter related to the chemoresponsiveness, but also could be a pure prognostic marker. TUBB3 expression levels differ by cell and tissue types. In some tissues, TUBB3 is constitutively expressed and is not inducible upon hypoxia. 21 In AGS cells, we observed TUBB3 was constitutively expressed in normoxia and became more induced in hypoxia.
The present study demonstrated that blockade of both VEGFR-1 and VEGFR-2 in conjunction with paclitaxel synergistically depresses induction of HIF-1α and TUBB3 expression, and more effectively increases cytotoxicity in gastric cancer cells.
tivation, and metastasis. Treatment with VEGFR-1 inhibitors, such as anti-VEGFR-1 antibody, suppresses tumor growth and metastasis in various models. 26 However, the degree of tumor growth inhibition varies. Some studies have reported that VEGFR-1 inhibition is not sufficient to block tumor growth without combined inhibition of VEGFR-2. 28,29 In addition, it is unclear if VEGFR-1 inhibitors, by preventing the binding of VEGF to VEGFR-1, increase the concentration of free VEGF that can subsequently activate VEGFR-2 and stimulate angiogenesis by an alternate pathway. The precise mechanisms of VEGFR-1 inhibitor functioning warrant further exploration. 26 We demonstrated that HIF-1α expressions increase in association with elevated PlGF. HIF-1α and TUBB3 were upregulated in hypoxia and became more induced in response to PlGF treatment. We also demonstrated that inhibition of VEGFR-1 is associated with decreases in HIF-1α and TUBB3 expression. Calvani, et al. 18 reported that VEGFR-2, rather than VEGFR-1, mediates VEGF-dependent induction of HIF-1α in hypoxic HCT116 colon cancer cells, suggesting that functional VEGFR-2, but not functional VEGFR-1, exists in this colon cancer cell line. Cell viability assay results demonstrated that AGS cell cytotoxicity was most pronounced when cells were treated simultaneously with paclitaxel, anti-VEGFR-1, and anti-VEGFR-2. Through these re- The cell viability assay demonstrated that AGS cell cytotoxicity was increased following combined treatment with paclitaxel and bevacizumab compared with treatment with paclitaxel alone. The increase of cytotoxicity was most pronounced when paclitaxel, anti-VEGFR-1, and anti-VEGFR-2 were applied simultaneously. Cells were maintained in normoxia or hypoxia for 24 h with paclitaxel following a 4-h pre-incubation with neutralizing antibody. Cell proliferation was normalized on the normoxic control. Values are expressed as means±SEM (n=3). *p<0.05. VEGFR, vascular endothelial growth factor receptor; SEM, standard error of the mean.