Cell Sublines Research Articles

Characterization of THLE-Cytochrome P450 (P450) Cell Lines: Gene Expression Background and Relationship to P450-Enzyme Activity

The hepatic SV40 large T-antigen immortalized human liver epithelial (THLE) cell line and sublines transfected with cytochromes P450 (P450s) are increasingly being used for evaluation of potential drug-induced liver injury. So far, the available information on transporter and enzyme expression in these transfected cell systems is scattered. The purpose of this study was to characterize THLE cell lines with respect to transporter and enzyme expression. The mRNA expression of 96 typical drug absorption, distribution, metabolism and excretion genes, which encode a selection of transporters, phase I and II drug-metabolizing enzymes, and nuclear hormone receptors, was investigated in five THLE cell lines transfected with individual human P450s and in mock-transfected THLE-null cells using real-time polymerase chain reaction. The majority of the analyzed genes was either absent or expressed at low levels in the THLE-null and THLE-P450 cells, apart from housekeeping genes and the individual transfected P450s. Enzyme activity measurements provided confirmatory functional data for CYP2C9 and CYP3A4. Comparison with gene expression in human liver revealed an overall much lower gene expression in the THLE cell lines. The low levels of expression of a broad range of P450 genes in the THLE cell lines highlight the value of studies undertaken with P450-expressing cell lines for investigation of mechanisms of P450 metabolite-mediated hepatotoxicity. However, when attempting to translate between data obtained in THLE cell lines in vitro and functional consequences in vivo, it is important to take account of their limited expression of genes encoding many other drug-metabolizing enzymes and hepatic transporters.

Low Androgen Sensitivity Is Associated With Low Levels of Akt Phosphorylation in LNCaP‐E9 Cells

We previously characterized LNCaP-E9, a low-androgen-sensitive LNCaP cell subline. LNCaP-E9 cells exhibit lower expression of androgen-regulated genes, including prostate-specific antigen (PSA), FK506 binding protein 5 (FKBP5), and prostatic acid phosphatase (PAcP), compared with LNCaP cells after treatment with the synthetic androgen R1881, confirming that the cells have low sensitivity to androgens. To understand the mechanism underlying low androgen sensitivity of LNCaP-E9 cells, we examined the activities of the Akt, p44/42, and p38 mitogen-activated protein kinase signaling pathways, all of which are known to be linked to androgen receptor signaling. We found that the phosphorylation of Akt at Ser473 was markedly lower in LNCaP-E9 cells than in LNCaP cells. Inhibition of Akt phosphorylation by the phosphatidylinositol 3-kinase inhibitor LY294002 resulted in reduction of PSA expression in LNCaP cells. Conversely, activation of Akt by serum starvation led to the induction of PSA expression in LNCaP-E9 cells. These results suggest that the impaired Akt phosphorylation in LNCaP-E9 cells is associated with low androgen sensitivity.

Functional Interplay between Caspase Cleavage and Phosphorylation Sculpts the Apoptotic Proteome

Caspase proteases are principal mediators of apoptosis, where they cleave hundreds of proteins. Phosphorylation also plays an important role in apoptosis, although the extent to which proteolytic and phosphorylation pathways crosstalk during programmed cell death remains poorly understood. Using a quantitative proteomic platform that integrates phosphorylation sites into the topographical maps of proteins, we identify a cohort of over 500 apoptosis-specific phosphorylation events and show that they are enriched on cleaved proteins and clustered around sites of caspase proteolysis. We find that caspase cleavage can expose new sites for phosphorylation, and, conversely, that phosphorylation at the +3 position of cleavage sites can directly promote substrate proteolysis by caspase-8. This study provides a global portrait of the apoptotic phosphoproteome, revealing heretofore unrecognized forms of functional crosstalk between phosphorylation and caspase proteolytic pathways that lead to enhanced rates of protein cleavage and the unveiling of new sites for phosphorylation.

In vitro cytotoxic activity of novel protoflavone analogs-selectivity against a multi-drug resistant cancer cell line

Protoapigenone (PA), a natural flavonoid possessing an unusual p-quinol moiety on its B ring, is a prospective novel anticancer lead compound currently in development, together with WYC0209, a potent synthetic PA analog. Previously, an increased cytotoxicity was found when a 3–4 carbon long aliphatic side-chain was present at C-1' for analogs of PA, while this was not the case when WYC0209 derivatives expressed the same moiety. Fifteen 1'-O-alkyl protoflavone derivatives were synthesized from genkwanin or 4'-hydroxy-6-methylflavone, thirteen of which are new compounds. All compounds, as well as PA, WYC0209 and fourteen of their previously reported analogs were also tested on a multi-drug resistant sub-cell line (MDR) of L5178 mouse T-cell lymphoma and on its parental counterpart (PAR). In general, derivatives expressing a free hydroxyl group at C-1' exerted the strongest activities, while C-1' substituted compounds were found much weaker. Derivatives of 6-methylflavone showed mild, but statistically significant selectivity against the MDR cell line. The results are in agreement with our previous findings for fundamental structure-activity relationships on protoflavones. 6-methylated protoflavones may serve as valuable leads for developing selective compounds against MDR cancer. Identical activity of other derivatives on the PAR and MDR cell lines suggests that cancer cells cannot confer resistance to protoflavones by ABCB1 efflux pump over-expression.

Significance of IL-6 in the transition of hormone-resistant prostate cancer and the induction of myeloid-derived suppressor cells

Hormone-resistant (HR) prostate cancers are highly aggressive and respond poorly to treatment. A better understanding of the molecular mechanisms involved in HR should lead to more rational approaches to therapy. The role of IL-6/STAT3 signaling in the transition of HR with aggressive tumor behavior and its possible link with myeloid-derived suppressor cells (MDSCs) were identified. In the present study, murine prostate cancer cell line (TRAMP-C1) and a hormone-resistant cell sub-line (TRAMP-HR) were used. Changes in tumor growth, invasion ability, and the responsible pathway were investigated in vitro and in vivo. We also examined the role of IL-6 in HR tumor progression and the recruitment of MDSCs. As seen in both in vitro and in vivo experiments, HR had aggressive tumor growth compared to TRAMP-C1. From mRNA and protein analysis, a higher expression of IL-6 associated with a more activated STAT3 was noted in HR tumor. When IL-6 signaling in prostate cancer was blocked, aggressive tumor behavior could be overcome. The underlying changes included decreased cell proliferation, less epithelial-mesenchymal transition, and decreased STAT3 activation. In addition to tumor progression, circulating IL-6 levels were significantly correlated with MDSC recruitment in vivo. Inhibition of IL-6 abrogated the recruitment of MDSCs in tumor- bearing mice, associated with slower tumor growth and attenuated angiogenesis. In conclusion, altered IL-6/STAT3 signaling is crucial in HR transition, aggressive behavior, and MDSC recruitment. These findings provide evidence for therapeutically targeting IL-6 signaling in prostate cancer.

Nuclear expression of Twist promotes lymphatic metastasis in esophageal squamous cell carcinoma

Twist-1 protein (also called Twist) has been suggested to be involved in tumor epithelial-mesenchymal transition (EMT) related progression, however, the mechanism by which twist promotes lymph node metastasis is not fully understood. In the present study, we found that nuclear twist expression is clearly correlated with lymph node (LN) metastasis as determined by immunohistochemistry (IHC). A highly invasive EC109 cell subline, EC109-P, was established by repeated in vitro transwell isolations for the cell model. Immunofluorescence (IF) assay demonstrated that nuclear twist expression was markedly higher in the highly invasive EC109-P cell line when compared with EC109 and EC9706 cells. Based on our cell model, the function and mechanism by which twist regulates LN metastasis in ESCC was investigated. The results showed that the overexpression of Twist could significantly increase the invasion and VEGF-C expression of EC9706 cells, whereas the knockdown of twist expression results in the opposite effects. This finding was further strengthened by the results of the analysis of co-expression of twist and VEGF-C by IHC in ESCC clinical samples. In summary, our study indicates that nuclear twist plays an important role in ESCC lymphatic metastasis by increasing the expression of VEGF-C. The combination of twist and VEGF-C detection could be a reliable prediction of LN metastasis in ESCC.

Significant Activity of Ecdysteroids on the Resistance to Doxorubicin in Mammalian Cancer Cells Expressing the Human ABCB1 Transporter

Multidrug resistance (MDR) is a major cause of failure of cancer chemotherapy. Fifty-eight ecdysteroids, herbal analogues of the insect molting hormone and their semisynthetic derivatives, were tested for their activity against L5178 mouse T-cell lymphoma cells (non-MDR) and their subcell line transfected with pHa MDR1/A retrovirus overexpressing the human ABCB1 efflux pump (MDR cell line). The compounds showed very low antiproliferative activities but modulated the efflux of rhodamine 123 mediated by the ABCB1 transporter. Roughly depending on the polarity, mild to strong synergism or antagonism was observed by combining ecdysteroids with doxorubicin, and specific structure-activity relationships were also found. Our results show the effect of ecdysteroids on MDR cancer cells for the first time. Less polar derivatives may serve as valuable leads toward a potent and safe resistance modulator. Biological significance of the resistance-increasing activity of the most abundant phytoecdysteroids including 20-hydroxyecdysone is yet to be clarified.

Epidermal growth factor receptor signaling pathway involved in progestin‐resistance of human endometrial carcinoma: In a mouse model

The aim of these investigations was to study the role of gefitinib (a specific oral epidermal growth factor receptor-tyrosine kinase inhibitor) on reversing progestin-resistance in a human endometrial carcinoma xenograft model. To study the effect of gefitinib and epidermal growth factor receptor (EGFR) overexpression on tumor progestin resistance, the Ishikawa endometrial carcinoma cell line was transfected to stably express a high level of EGFR, which resulted in the progestin-resistant Ishikawa-pLWERNL subcell line. BALB/c nude mice were injected subcutaneously with the parental Ishikawa cell line and the Ishikawa-pLWERNL cell line. Therapy experiments with gefitinib alone or in combination with medroxyprogesterone acetate (MPA) were done and samples were analyzed for EGFR and progesterone receptor isoform B (PR-B) expression by Western blot and immumohistochemistry analyses. Role in blocking EGFR autophosphorylation and its downstream signaling pathway and antagonizing progestin resistance by gefitinib was investigated by Western blot analysis. EGFR expression was higher in progestin-resistant Ishikawa-pLWERNL endometrial cancer (EC) xenografts than in progestin-sensitive Ishikawa EC xenografts; in contrast, PR-B was higher in Ishikawa xenografts than in Ishikawa-pLWERNL xenografts. Higher EGFR expression reduced sensitivity to progestin and decreased PR-B expression in Ishikawa xenografts; it also abnormally activated EGFR autophosphorylation and its downstream signaling pathway. Gefitinib effectively inhibited the proliferation of EC xenografts that overexpressed EGFR, and reversed hormone resistance in progestin-resistant EC xenografts. The present study describes an in vivo model that can provide a valuable tool in studying the interaction of overexpressed EGFR and progestin resistance in EC. Gefitinib may be useful in the treatment of progestin-resistant EC.

Abstract B28: Downregulation of vimentin confers resistance to the microtubule stabilizing agents peloruside A and laulimalide in 1A9 human ovarian carcinoma cells

Abstract Microtubule-targeting agents are an important class of drugs for the treatment of cancer. Although the development of drug resistance in cancer cells often limits the clinical efficacy of these agents, the mechanisms of resistance are not fully understood. Peloruside A (PLA) and laulimalide (LAU) are two marine sponge-derived stabilizing agents of microtubules. PLA and LAU represent the only known class of non-taxane site binding microtubule stabilizing agents and have a number of clinically useful potential advantages over paclitaxel, a widely used tubulin-targeting anticancer drug, including reduced susceptibility to the multidrug resistance P-glycoprotein efflux pump, good efficacy against taxane-resistant cells with mutations in the taxoid site, and more effective antitumor effects in non-small cell lung tumor xenografts. To understand the mechanisms of resistance to PLA and LAU, we used two drug-resistant sublines of the 1A9 human ovarian carcinoma cell line, R1 and L4. R1 cells are 6-fold resistant to PLA but remain fully sensitive to LAU; whereas, the L4 cells are 39-fold resistant to both PLA and LAU. We previously showed that both resistant cell lines exhibit different single point-mutations in their βI-tubulin gene. The L4 cells also have a high abundance of βII- and βIII-tubulin isotypes, which play a partial role in the resistance phenotype. To determine other protein changes that might contribute to the resistant phenotype of the R1 and L4 cells, we examined the proteome of the resistant cells using two-dimensional differential in-gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The proteomic analysis identified several proteins that were significantly altered relative to the parental 1A9 cells, including a decreased expression of vimentin in L4 cells. Using Western blotting and immunocytochemistry, the decrease in vimentin abundance seen in 2D-DIGE was confirmed. Vimentin levels were unchanged in the PLA-resistant R1 cells as well as two paclitaxel/epothilone-resistant sublines of 1A9 cells. Transfection with a full-length human vimentin cDNA partially restored sensitivity of L4 cells to PLA and LAU by 23% and 17% respectively. In vimentin-expressing parental 1A9 cells, tubulin-targeting and DNA-damaging agents caused degradation of vimentin and generation of vimentin degradation products. In conclusion, this study is the first to demonstrate a novel function for vimentin as a cellular determinant of the response to microtubule stabilizing agents, and that this effect is likely to be associated with the mode of cell killing by these agents. Since many of the proteins identified in this study have not previously been shown to play a role in drug resistance, our results also introduce potential new mechanisms by which cancer cells may become resistant to antimicrotubule agents.

Abstract A52: Comparison of the efficacy of different mTOR inhibitors for the treatment of prostate cancer

Abstract Background: We previously showed that the mTOR pathway is activated in castration resistant prostate cancer (CRPC) cell lines while its inhibition resulted in upregulation of androgen receptor (AR) signaling, resulting in resistance to mTOR-based therapy (Wang et al., 2008, Oncogene, 27(56):7106-17). mTOR forms two complexes: activation of mTORC1 (mTOR, raptor) increases mRNA translation by phosphorylation of the downstream molecules p70-S6K (S6K) and eIF4E binding protein 1 (4E-BP1), while activation of mTORC2 (mTOR, rictor) leads to activation of Akt, which is also activated by phosphatidylinositol 3-kinase (PI3K). A number of drugs targeting these complexes are currently available, among them, RAD001, which inhibits mTORC1 alone, BEZ235, a dual mTORC1/PI3K inhibitor, and Torin1, an ATP-competitive inhibitor which prevents activation of both mTORC1 and mTORC2. The goal of the present study was to identify the drug that is most effective against PCa either alone or in combination with the AR inhibitor bicalutamide. Results: The drugs were used in a number of cell lines including C4-2 and LNCaP-AI, two CRPC sublines of androgen-dependent LNCaP cells, as well as CWR-R1 and CWR22Rv1, two lines derived from relapsed CWR22 tumors in castrated mice. Another androgen-sensitive cell line, PC-346C, was also used. RAD001 dose responsibly inhibited growth in C4-2 cells, but the other cell lines were resistant to this therapy, either alone or in combination with bicalutamide. In contrast, LNCaP-AI and CWR-R1 cells were also sensitive to BEZ235 and Torin1, but PC-346C cells were resistant to all three. Hence we investigated the molecular mechanism underlying the resistance of these cells to mTOR inhibitors. Clonogenic assays revealed that 20 nM BEZ235 and 25 nM Torin1 are optimal doses that prevent growth of PCa cells, effect comparable of that of 10 nM of RAD001 (physiological concentration in blood of patients under treatment with this drug). Comparison of the downstream targets of the various drugs at these doses revealed that 4E-BP1 and Akt are highly phosphorylated in both C4-2 and PC-346C cells despite RAD001 treatment, whereas BEZ235 and Torin1 inhibited 4E-BP1 phosphorylation. This is despite the fact that AR levels, but not AR transcriptional activity, increased in BEZ235 treated cells, but not in those treated with RAD001 and Torin1. However, phosphorylation of its binding partner eIF4E increased in PC-346C cells but not in C4-2 cells. Conclusion: Phosphorylation of 4E-BP1 and eIF4E, as well as Akt, needs to be downregulated in order for the drug to be effective. The effects of BEZ235 are likely complicated by the increase in AR levels despite the use of bicalutamide. Therefore, a stronger AR inhibitor, such as MDV3100, in combination with this drug, may be required. Acknowledgments: We would like to thank Dr. David Sabatini for the drug Torin1, Novartis Pharmaceuticals for providing BEZ235 and RAD001, and Astra Zeneca, Cheshire, UK, for the drug bicalutamide. CWR-R1 cells were generously provided by Dr Elizabeth Wilson (University of North Carolina), while PC-346C cells were from Dr. W.M. van Weerden, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands.

Insufficient Radiofrequency Ablation Promotes Angiogenesis of Residual Hepatocellular Carcinoma via HIF-1α/VEGFA

BackgroundThe mechanism of rapid growth of the residual tumor after radiofrequency (RF) ablation is poorly understood. In this study, we investigated the effect of hyperthermia on HepG2 cells and generated a subline with enhanced viability and dys-regulated angiogenesis in vivo, which was used as a model to further determine the molecular mechanism of the rapid growth of residual HCC after RF ablation.Methodology/Principal FindingsHeat treatment was used to establish sublines of HepG2 cells. A subline (HepG2 k) with a relatively higher viability and significant heat tolerance was selected. The cellular protein levels of VEGFA, HIF-1α and p-Akt, VEGFA mRNA and secreted VEGFA were measured, and all of these were up-regulated in this subline compared to parental HepG2 cells. HIF-1α inhibitor YC-1 and VEGFA siRNA inhibited the high viability of the subline. The conditioned media from the subline exerted stronger pro-angiogenic effects. Bevacizumab, VEGFA siRNA and YC-1 inhibited proangiogenic effects of the conditioned media of HepG2 k cells and abolished the difference between parental HepG2 cells and HepG2 k cells. For in vivo studies, a nude mouse model was used, and the efficacy of bavacizumab was determined. HepG2 k tumor had stronger pro-angiogenic effects than parental HepG2 tumor. Bevacizumab could inhibit the tumor growth and angiogenesis, and also eliminate the difference in tumor growth and angiogenesis between parental HepG2 tumor and HepG2 k tumor in vivo.Conclusions/SignificanceThe angiogenesis induced by HIF1α/VEGFA produced by altered cells after hyperthermia treatment may play an important role in the rapid growth of residual HCC after RF ablation. Bevacizumab may be a good candidate drug for preventing and treating the process.

Abstract A12: Identification of Nrdp1 as a novel androgen receptor transcription target differentially regulated in androgen-dependent and independent prostate cancer

Abstract Background: The ErbB receptor tyrosine kinase family regulates proliferation and survival in prostate cancer (PCa). We recently showed that ErbB3 plays a significant role in increasing androgen receptor (AR) transcriptional activity and in causing castration-resistant PCa (CRPC). Reciprocally, AR maintained castration sensitivity by suppressing ErbB3 levels through transcriptional regulation of E3 ubiquitin ligase Nrdp1, while loss of AR regulation of Nrdp1 resulted in an unrestricted surge in ErbB3 levels, and cell growth. Here, we investigate whether Nrdp1 is a transcriptional target of the AR in PCa cells. Results: The promoter region of Nrdp1 contains three androgen response elements (AREs) - one located 215 aa upstream of the transcriptional start site (ARE.03), and two within an internal promoter (ARE.01 and ARE.02). Chromatin immunoprecipitation (ChIP) studies revealed AR binding in PCa cells to ARE.03. AR binding to ARE.03 was found to be androgen regulated in androgen-dependent LNCaP cells, whereas no AR binding to ARE.01 or ARE.02 has been detected in this cell line. Luciferase assay to determine AR transcriptional activity on ARE.03 showed significant response to androgens, whereas full mutation of ARE.03 abolished AR transcriptional activity. However, the AR failed to bind to ARE.03 in C4-2 and LNCaP-AI cell lines, CRPC sublines of LNCaP cells, although no mutations in these regions were identified, while AR continued to bind the AR to PSA ARE in these cells, indicating that the AR was still active. Thus the AR binds to PSA ARE in both androgen-dependent and –independent cells, but to ARE.03 only in androgen-dependent but not –independent cells. Luciferase assay in LNCaP-AI cells showed decreased AR transcriptional activity on ARE.03 when compared to LNCaP, supporting decreased AR binding to this region. Hence we investigated the cause for the differential binding of the AR in these two areas. The structural protein Filamin A (FlnA) was earlier shown to cleave to a 90 kDa fragment (FlnA16-24) whose nuclear localization maintained androgen dependence. We investigated whether this protein played a role in regulation of AR binding to ARE.03 in PCa cells. Transfection of FlnA16-24 in C4-2 cells resulted in localization of FlnA 16-24 to the nucleus, and restored AR binding to ARE.03. FlnA 16-24 significantly increased luciferase activity on ARE.03, while suppressing AR activity on PSA indicating that this AR binding protein is required for AR-mediated transcription of Nrdp1. Conclusions: Our results indicated that Nrdp1 is regulated in androgen sensitive cells, but not in CRPC cells, by AR binding to ARE.03, whereas no such difference in AR binding to PSA ARE exists. We show that AR binding to ARE.03 is abolished in CRPC cells because of a decreased expression of nuclear FlnA, and that restoration of nuclear FlnA will restore androgen-sensitive cell growth. Our data indicate that the AR-coregulator FlnA is required for AR binding to the Nrdp1 promoter and that loss of this protein results in the failure of AR to regulate Nrdp1 transcription, resulting in unrestricted increase in ErbB3. These data identified Nrdp1 as a novel target of AR transcriptional activity in androgen-dependent but not in CRPC cells.

Abstract 3465: The effect of ephrin-A1 on the resistance of photodynamic therapy in esophageal cancer cells

Abstract Photodynamic therapy (PDT) has been demonstrated as an effective minimally invasive treatment modality for early esophageal cancer. However, the molecular action in esophageal cancer during PDT is hardly known. To know better about the mechanisms of the resistance to PDT in esophageal cancer cells, we selected the PDT-resistant sub-cell lines and investigated the global gene modulations in the resistant cells by whole-genome microarray. Photofrin is a most frequently used photosentizer in PDT. We have selected two Photofrin-PDT resistant sub-cell lines, PDTR4-4 and PDTR4-6, derived from CE48T/VGH (CE48T) esophageal cancer cell line. At least 700 genes reached to over 1.5 fold changes in both subcell lines compared to parental cell line. Among the candidate genes, TNF and EFNA1 genes were notably up-regulated in both resistant cell lines and down-regulated in Photofrin-PDT treated cells compared to untreated cells by microarray analysis. It revealed that TNF and EFNA1 encoded proteins may play some roles to promote the resistance to Photofrin-PDT. EFNA1 encodes ephrin-A1, a ligand belongs to a receptor tyrosine kinase. It has been found associated with tumor formation and up-regulated in various cancer types. TNF encodes tumor necrosis factor-alpha (TNF-alpha)cytokine. Because TNF-alpha can induce ephrin-A1 expression in endothelial cells, we suggest TNF-alpha may activate the expression of ephrin-A1 and induce the resistance to Photofrin-PDT in esophageal cancer cells. Currently, we investigated whether TNF-alpha can activate the expression of ephrin-A1 in esophageal cancer cells. We clearly observed recombinant TNF- alpha can activate the gene expression of EFNA1 in transcription level. To further demonstrate Photofrin-PDT inhibit the gene expression of TNF and EFNA1 in esophageal cancer cells, we detected the mRNA expression of both TNF and EFNA1 in CE48T cells upon Photofrin-PDT treatment by quantitative PCR. We clearly observed Photofrin-PDT can inhibit the expression of the transcripts encoded by both TNF and EFNA1. The protein expression of ephrin-A1 was also suppressed by Photofrin- PDT. Meanwhile, we determined the background expression levels of both TNF and EFNA1 in PDTR4-6 and another more resistant sub-cell line PDTR5-8. Compared to parental cells, both PDTR4-6 and PDTR5-8 displayed significant over-expression of TNF and EFNA1 in transcription levels. Finally, we investigated whether exogenous ephrin-A1 can enhance the resistance to Photofrin-PDT in esophageal cancer cells. We preliminarily observed the CE48T cells which incubated in the medium containing 10 and 25 ng/ml of recombinant ephrin-A1 were more resistant to Photofrin-PDT induced cell death by around 20 % versus untreated cells. Based on our findings, we suggest TNF-alpha -induced ephrin-A1 expression may contribute to Photofrin-PDT resistance in esophageal cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3465. doi:1538-7445.AM2012-3465

Abstract 1904: Transforming growth factor-beta mediated epithelial to mesenchymal transition contributes to in vivo resistance to sorafenib in hepatocellular carcinoma

Abstract Sorafenib, a multikinase inhibitor targeting Raf kinase and vascular endothelial growth factor receptor, is currently the only approved agent for hepatocellular carcinoma (HCC). However, the majority of HCC patients are inherently resistant to sorafenib treatment. To study molecular mechanisms underlying the resistance to sorafenib in vivo, we studied immunocompromised mice implanted with xenografts of human HCC cells subcutaneously, and identified tumors that showed primary resistance to sorafenib. Primary cultured cells were established from sorafenib-resistant xenografts, and verified for in vitro and in vivo sensitivity to sorafenib. The differentially expressed genes of the resistant cells versus control cells were analyzed by cDNA microarray. The expressions of candidate markers were confirmed by Western blotting, and their significances were verified by specific pathway inhibitors. A subline of Huh7 cells was established by primary culture from a sorafenib-resistant HCC-xenograft (designated as Huh7.1.SR). Compared to Huh7 cells primarily cultured from HCC-xenograft treated with vehicle alone (designated as Huh7.1.v), Huh7.1.SR had a moderate increase of resistance to sorafenib in vitro, with 50%-inhibitory concentration (IC50) increased from 5 to 10 μM. Subcutaneous xenograft studies confirmed that Huh7.1.SR retained the phenotype of in vivo resistance to sorafenib. The IPA (ingenuity pathway analysis) of differentially expressed genes of Huh7.1.SR versus Huh7.1.v cells revealed several pathway networks centering at transforming growth factor beta receptor (TGFBR), hepatocyte nuclear factors, NOTCH, pro-inflammatory cytokines, and nuclear factor kappa B. Huh7.1.SR had an increased expression of TGF-beta and p-Smad2, indicating that the TGF-beta pathway was activated. Huh7.1.SR cells expressed phenotypic features of epithelial to mesenchymal transition (EMT), including higher expression of vimentin, lower expression of E-cadherin, and increased cell migration. The expressions of Slug and Twist, two well-known transcriptional factors of inducing EMT, also increased in Huh7.1.SR cells. Treatment of SD-208, an inhibitor of TGF-beta receptor, partially reversed the phenotypic features of EMT in Huh7.1.SR cells. Moreover, concomitant treatment of SD-208 improved the sensitivity to sorafenib in Huh7.1.SR cells. Our data indicate that activation of TGF-beta signaling pathway is an important mechanism mediating the resistance to sorafenib in HCC in vivo. (The study was supported by the grant NSC 100-2325-B-002-043- from National Science Council of Taiwan) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1904. doi:1538-7445.AM2012-1904

Abstract 4324: Axl receptor tyrosine kinase expression regulates tumor cell invasion and migration in colorectal cancer

Abstract Background Colorectal cancer (CRC) is the third most common cancer in the UK with around 80% of patients undergoing surgery followed by adjuvant chemotherapy treatment. Among patients with clinicopathologically defined poor stage II and stage III CRC, there are subsets of patients who will not benefit from adjuvant 5-FU or 5-FU/oxaliplatin treatment. In fact, there is preclinical evidence to suggest that some of these patients may actually do worse when treated with adjuvant chemotherapy. The aim of this study was to develop in vitro adjuvant colon cancer models with the ultimate goal to identify novel treatment strategies for early stage CRC. Method Progressively invasive populations from parental HCT116 were generated using invasion chambers. Analysis of migration and invasion was carried out using the XCELLigence system, protein activity/expression using receptor tyrosine kinase array (RTK) and Western blotting, colony forming ability was assessed by survival assays and drug sensitivity using MTT assay and Flow cytometry. Results Six sublines of HCT116 cells were initially generated (I1-6) which displayed an increasing invasive/migratory phenotype compared to the parental cell line. At the molecular level, we found increases in basal activity of a number of RTK in the invasive sublines, such as Axl (a phenomenon which is characterized with a more aggressive nature in cancers) and also in other kinases such as ERK1/2, Akt and STAT3. Decreased activation of the EGFR and HER2 receptors was also noted in the invasive cell lines. In addition, exposure to sub-lethal 5-FU(IC20) doses resulted in significant increases in migration in these cell line models. Silencing of Axl resulted in significant decreases in migration of the invasive subpopulations even in the presence of 5-FU. Colony forming assays highlighted an increased ability to form colonies in the invasive sub-populations which also displayed drug resistance to both 5-FU and Oxaliplatin. The generation of invasive cell lines was also carried out in a number of other cell lines which confirmed that an increase in Axl expression could increase the migratory phenotype regardless of the genetic background of the cell and also that the results obtained are not an artefact of the cell line we have used. Conclusion Using our in-house developed in vitro preclinical cell line model we have begun to characterise the changes which tumour cells appear to go through during the transition to an invasive phenotype. We have highlighted Axl as a potential target for inhibition in early stage colorectal cancer as increases in total Axl protein levels correlate well with increases in invasive capacity in our models and silencing of the protein expression by siRNA results in ablation of the invasive potential in our model and also in a panel of CRC cell lines. Combination of an Axl small molecule inhibitor could potentially be a novel treatment strategy for early stage CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4324. doi:1538-7445.AM2012-4324

Abstract 2731: Cetuximab resistance is associated with increased dependence on lipogenesis

Abstract Sterol regulatory element binding proteins (SREBPs) are key transcriptional regulators of cellular lipid homeostasis. By regulating the maturation of SREBPs through the PI3K/Akt and MEK/Erk signaling pathways, the epidermal growth factor receptor (EGFR) regulates de novo lipogenesis, which is essential for the structural needs of proliferation of cancer cells. The objective of current study is to determine the role of inhibition of lipogenesis by cetuximab, an FDA-approved EGFR-blocking antibody, in cetuximab-induced antiproliferative/antisurvival effects in cancer cells and to identify new combinations of cetuximab with approaches interfering with lipid metabolism for improved therapy. We found that addition of cetuximab into culture medium inhibited the activation of SREBP-1 (appearance of a nuclear form through cleavage of SREBP-1 precursor) in HN5 head and neck cancer cells but not in a cetuximab-resistant subline of HN5 cells (HN5R). Compared with parental HN5 cells, HN5R cells exhibit remarkably increased sensitivity to treatment with two pharmacological lipogenesis inhibitors, C75 (a fatty acid synthase inhibitor) and TOFA (an acetyl-CoA carboxylase inhibitor), suggesting that the cetuximab-resistant cells are more dependent on de novo fatty acid synthesis for survival, and combination of cetuximab with lipogenesis inhibitors may be a novel combination therapy. On the other hand, the increased dependence of cancer cells on lipogenesis suggests that cetuximab-resistant cells might also be sensitive to agents that promote fatty acid α-oxidation. We found treatment of HN5R cells with fenofibrate, an agonist of peroxisome proliferator-activated receptor alpha that transcriptionally activates the genes for inducing fatty acid α-oxidation, disrupted the redox balance in the cetuximab-resistant cells, leading to increased production of ROS and depolarization of the mitochondrial membrane potential that resulted in apoptosis. In summary, our data indicate that the antiproliferative/antisurvival effects of cetuximab in cancer cells depend on effective inhibition of lipogenesis. Pharmacological approaches that forcibly switch cancer cell metabolism from aerobic glycolysis to fatty acid α-oxidation may overcome cetuximab resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2731. doi:1538-7445.AM2012-2731

Abstract 5306: Overexpression of RAD51 promotes brain metastases from breast cancer

Abstract Overexpression of the DNA double-strand break (DSB) repair gene RAD51 was identified as part of a 13-gene signature predictive of rapid brain metastasis development (≤3 years vs. >3 years after diagnosis, HR=3.03, p=0.0008) in primary tumors of chemotherapy-naïve HER2+ metastatic breast cancer patients. We also found RAD51 mRNA overexpressed (1.5-fold, p=0.001) in resected brain metastases compared to primary breast tumors from the same patients. In addition, RAD51 expression was significantly higher (1.4-fold, p=0.008,) in brain metastases compared to systemic (bone/lung) metastases. We therefore hypothesize that RAD51 promotes brain metastasis development in breast cancer patients. Given the known role of RAD51 in homologous recombination, important for the repair of DNA DSBs, it was unknown whether RAD51 would promote brain metastasis of breast cancer only in the presence of DNA-damaging chemotherapy or, alternatively, have endogenous biological functions in tumor cells. To functionally evaluate whether RAD51 promotes brain metastases, RAD51 was overexpressed at physiologically relevant levels in a brain-metastatic variants of triple-negative MDA-MB-231 breast carcinoma (231-BR) cells. In vitro, this resulted in a ∼30% increase in clonogenic colony formation (p=0.004), without affecting cell proliferation rates. RAD51 overexpression also conferred chemoresistance to DNA DSB inducing agents such as 25uM carboplatin (p=0.03) and 25nM doxorubicin (p=0.02), but not to agents that do not cause DNA DSBs, such as 2.5nM paclitaxel. In vivo, overexpression of RAD51 in 231-BR cells increased formation of large brain metastases, the equivalent of clinically detectable lesions, (4.0 and 3.3-fold, p=0.01 and p=0.005) and micro-metastases (3.1 and 3.5-fold, p=0.01 and p=0.001) in two independent xenograft experiments. Importantly, this brain-metastasis promoting effect occurred without drugs to induce DSB. This effect was recapitulated in a second animal model based on a brain-metastatic subline of 4T1 murine mammary carcinoma cells (4T1-BR). In this model, RAD51 overexpression increased large brain metastases (3.3-fold, p=0.02) and micro-metastases (2.0-fold, p=0.04) compared to vector control. ShRNA-mediated RAD51 knockdown in 4T1-BR cells resulted in the opposite effect, reducing large brain metastases (1.8-fold, p=0.1) and micro-metastases (2.5-fold, p=0.04). Together these data suggest a driving role for RAD51 in the development of brain metastases from breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5306. doi:1538-7445.AM2012-5306

Abstract 1292: Mutually exclusive expression of SOX2 and SOX9 in lung adenocarcinoma cells and its implications

Abstract SOX family genes play critical roles in embryonic development, cell fate determination, differentiation, and proliferation. Some members of the SOX family, including SOX2 and SOX9, have been shown as hall marks of lung cancers. However, their roles in lung cancer formation or progression still need to be explored. Here we show an unique expression pattern of SOX2 and SOX9 in lung cancer cells. Using a series of previously established lung adenocarcinoma cell sub-lines which were sequentially selected for increasing cell invasiveness, we found that SOX9 was abundant in highly tumorigenic and invasive sub-lines whereas absent in less tumorigenic and low invasive ones. Interestingly, SOX2 and SOX9 were mutually exclusively expressed among those sub-lines. Forced expression and gene silencing experiments in those cell lines indicated that, while SOX9 is important for cancer cell proliferation, SOX2 is involved in regulation of cell invasion. The involvoed molecular pathways subject to regulations by SOX9 and SOX2 have been partially elucidated and will be demonstrated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1292. doi:1538-7445.AM2012-1292

Abstract LB-343: Defining mechanisms of endocrine resistance in breast cancer using whole exome sequencing

Abstract About 70% of breast cancers are estrogen receptor positive (ER+) and current treatments target either ER activity (Tamoxifen and Fulvestrant) or the production of estrogen (aromatase inhibitors). These treatments have led to significant increases in disease-free and overall survival, but after initial response to these agents, many ER+ cancers progress to endocrine resistance. There have been several attempts to understand the molecular mechanisms that lead to acquired resistance, but a clear understanding is still lacking. We hypothesize that endocrine resistant cells in the tumor arise by acquiring compensatory somatic mutations, and that some of these driver mutations responsible for resistance are in oncogenes or tumor suppressor genes. To test this we performed whole exome sequencing on three of MCF7 breast cancer cell sublines that are resistant to either tamoxifen, fulvestrant or exemestane - drugs commonly used in the clinic to treat ER+ breast cancers. An Illumina TruSEQ adaptor-based exonic library was prepared using genomic DNA from the resistant and control cell lines, and multiplexed on an Illumina HiSeq 2000 sequencer (100bp paired-end reads). Reads were aligned to human genome build 19 using BWA, variants were called using SAMTools, somatic mutations in endocrine cell lines were determined with VarScan, and amino acid changes were annotated using ANNOVAR. We have identified several non-synonymous mutations in cancer-relevant genes and are currently working to validate these targets. Studying these genes will increase our understanding of how tumors overcome the requirement for estrogen signaling and ultimately, we hope that comparing tumor profiles of patients with endocrine resistance disease will lead to therapeutics targeted to these secondary mutational lesions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-343. doi:1538-7445.AM2012-LB-343

Abstract 1233: A triad of Brk, HER2 and STAT3 in regulating epithelial and mesenchymal transition and survival of breast cancer cells

Abstract Breast tumor kinase (Brk) is a nonreceptor protein-tyrosine kinase recently shown to be coexpressed with human epidermal growth factor receptor-2 (HER2) at high levels in tumor specimens from patients with invasive ductal breast cancer. Brk is associated with HER2 in breast cancer cells and can directly phosphorylate signal transducers and activators of transcription-3 (STAT3). However, the biological functions of these interactions remain largely unknown. In current study, we developed a subline from HER2-transfected MCF7 breast cancer cells through in vivo selection in nude mice. Overexpression of HER2 led to a higher level of Brk in MCF7 cells and the in vivo selection resulted in a subline with markedly enhanced epithelial and mesenchymal transition (EMT) phenotype and markedly increased migration potential. We named the subline cells EMT1. Both the EMT phenotype and survival of the EMT1 cells heavily depend on Brk, because knockdown of Brk through RNA interference reversed the EMT phenotype, reduced cell migration potential and induced cell death via apoptosis in the EMT1 cells. We further found that the level of activation-specific phosphorylated STAT3 was markedly increased in the EMT1 cells compared with control vector-transfected or HER2-transfected MCF7 cells. Knockdown of Brk significantly reduced the level of STAT3 phosphorylation in the EMT1 cells. Overexpression of a constitutively active STAT3 counteracted Brk knockdown-induced inhibition of cell migration potential and Brk knockdown-induced apoptosis in the EMT1 cells. Together, these preliminary findings support a new paradigm for regulating breast cancer cell EMT and survival that involves interaction and coordination between Brk, HER2, and STAT3. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1233. doi:1538-7445.AM2012-1233