Alba Ginés

Pharmacogenomic approach for the identification of novel determinants of acquired resistance to oxaliplatin in colorectal cancer

Oxaliplatin is a third-generation platinum agent used in colorectal cancer treatment. Oxaliplatin resistance acquisition is a complex process mainly based on alteration of genes and pathways involved in its mechanism of action. Therefore, our purpose was to perform a gene expression screening in an in vitro model to identify genes that could play a role in oxaliplatin resistance acquisition processes. Four colorectal cancer cell lines and their oxaliplatin-resistant derived sublines were compared. Microarray analysis was done using Human 19K Oligo Array Slides. RNA from cells were hybridized with a commercial RNA reference sample and labeled with both fluorochromes Cy3 and Cy5. Data were analyzed by hierarchical clustering method. Subsequently, quantitative real-time PCR (qRT-PCR) was used to corroborate microarray data, considering as positively validated those genes that showed significant differences in expression levels between groups and a correlation between microarray and qRT-PCR data. By microarray analysis, 32 candidate genes were identified. After validation process by qRT-PCR, the genes AKT1, CDK5, TRIP, GARP, RGS11, and UGCGL1 were positively validated. The 3 first genes proved to be involved in regulation of nuclear factor-κβ antiapoptotic transcription factor previously related to drug resistance, and the other 3 genes are novel finds. We have identified 6 genes related to oxaliplatin resistance acquisition. These findings are of paramount importance to understand these processes better and open new lines of study to elucidate the relevance of this pharmacogenomic approach into the clinic. [Mol Cancer Ther 2009;8(1):194–202]

A proteomic approach links decreased pyruvate kinase M2 expression to oxaliplatin resistance in patients with colorectal cancer and in human cell lines

We aimed to gain further understanding of the molecular mechanisms involved in oxaliplatin resistance in colorectal cancer by using a proteomic approach. A 5-fold oxaliplatin-resistant cell line, HTOXAR3, was compared with its parental cell line, HT29, using two-dimensional PAGE. Mass spectrometry, Western blot, and real-time quantitative PCR confirmed the down-regulation of pyruvate kinase M2 (PK-M2) in HTOXAR3 cells. In a panel of eight colorectal cancer cell lines, we found a negative correlation between oxaliplatin resistance and PK-M2 mRNA levels (Spearman r = −0.846, P = 0.008). Oxaliplatin exposure in both HT29 and HTOXAR3 led to PK-M2 mRNA up-regulation. PK-M2 mRNA levels were measured by real-time quantitative PCR in 41 tumors treated with oxaliplatin/5-fluorouracil. Tumors with the lowest PK-M2 levels attained the lowest response rates (20% versus 64.5%, P = 0.026). High PK-M2 levels were associated with high p53 levels (P = 0.032). In conclusion, the data provided clearly link PK-M2 expression and oxaliplatin resistance mechanisms and further implicate PK-M2 as a predictive marker of response in patients with oxaliplatin-treated colorectal cancer.[Mol Cancer Ther 2009;8(4):771–8]

Increased levels of copper efflux transporter ATP7B are associated with poor outcome in colorectal cancer patients receiving oxaliplatin-based chemotherapy.

Recently, the copper efflux transporters ATP7B and ATP7A have been implicated in the transport of and resistance to platinum drugs in breast and ovarian cancers. Because of the extensive use of oxaliplatin in colorectal cancer (CRC), we examined the expression of both transporters in tumors from CRC patients treated with oxaliplatin/5FU and sought to determine whether their expression can predict clinical outcome in these patients. ATP7B and ATP7A levels were determined by quantitative real‐time PCR in 50 primary tumors of previously untreated patients with advanced colorectal adenocarcinoma who were subsequently treated with oxaliplatin/5FU. Additionally, ATP7B protein expression was assessed by immunohistochemical staining using a tissue microarray. Patients with the lowest mRNA expression levels of ATP7B had a significantly longer time to progression (TTP) (p = 0.0009) than patients with the highest levels (12.14 months vs. 6.43 months) who also had an increased risk of progression (HR = 3.56; 95% CI, 1.6–7.9; p = 0.002). Furthermore, patients with low levels of both protein and mRNA of ATP7B derived the maximum benefit from oxaliplatin/5FU with the longest TTP as compared with patients with high levels of ATP7B protein and mRNA (14.64 months vs. 4.63 months, respectively, p = 0.01) and showed a nonsignificant trend toward a lower response rate (37.5% and 75%, respectively). In conclusion, ATP7B mRNA and protein expression in colorectal tumors is associated with clinical outcome to oxaliplatin/5FU. Prospective studies are required to evaluate the role of this marker in tailoring chemotherapy.

Tumor-Related Molecular Mechanisms of Oxaliplatin Resistance

Oxaliplatin was the first platinum drug with proven activity against colorectal tumors, becoming a standard in the management of this malignancy. It is also considered for the treatment of pancreatic and gastric cancers. However, a major reason for treatment failure still is the existence of tumor intrinsic or acquired resistance. Consequently, it is important to understand the molecular mechanisms underlying the appearance of this phenomenon to find ways of circumventing it and to improve and optimize treatments. This review will be focused on recent discoveries about oxaliplatin tumor-related resistance mechanisms, including alterations in transport, detoxification, DNA damage response and repair, cell death (apoptotic and nonapoptotic), and epigenetic mechanisms. Mol Cancer Ther; 14(8); 1767–76. ©2015 AACR.

PKM2 Subcellular Localization Is Involved in Oxaliplatin Resistance Acquisition in HT29 Human Colorectal Cancer Cell Lines

Chemoresistance is the main cause of treatment failure in advanced colorectal cancer (CRC). However, molecular mechanisms underlying this phenomenon remain to be elucidated. In a previous work we identified low levels of PKM2 as a putative oxaliplatin-resistance marker in HT29 CRC cell lines and also in patients. In order to assess how PKM2 influences oxaliplatin response in CRC cells, we silenced PKM2 using specific siRNAs in HT29, SW480 and HCT116 cells. MTT test demonstrated that PKM2 silencing induced resistance in HT29 and SW480 cells and sensitivity in HCT116 cells. Same experiments in isogenic HCT116 p53 null cells and double silencing of p53 and PKM2 in HT29 cells failed to show an influence of p53. By using trypan blue stain and FITC-Annexin V/PI tests we detected that PKM2 knockdown was associated with an increase in cell viability but not with a decrease in apoptosis activation in HT29 cells. Fluorescence microscopy revealed PKM2 nuclear translocation in response to oxaliplatin in HCT116 and HT29 cells but not in OXA-resistant HTOXAR3 cells. Finally, by using a qPCR Array we demonstrated that oxaliplatin and PKM2 silencing altered cell death gene expression patterns including those of BMF, which was significantly increased in HT29 cells in response to oxaliplatin, in a dose and time-dependent manner, but not in siPKM2-HT29 and HTOXAR3 cells. BMF gene silencing in HT29 cells lead to a decrease in oxaliplatin-induced cell death. In conclusion, our data report new non-glycolytic roles of PKM2 in response to genotoxic damage and proposes BMF as a possible target gene of PKM2 to be involved in oxaliplatin response and resistance in CRC cells.

Abstract 5478: Curcumin mediates reversion to oxaliplatin-acquired resistance in colorectal cancer cell lines through modulation of nuclear factor κB (NFκB) and cyclin-dependent kinase 5 (CDK5)

Background: In previous work we have observed hyper-activation of the transcription factor (TF) NF-κB and an up-regulation of the Cyclin-dependent kinase 5 (CDK5) in human colorectal cancer cell lines with acquired resistance to oxaliplatin (OXA). Moreover, OXA treatment induced NF-κB activation. Curcumin (diferuloylmethane, CURC), the major active ingredient of turmeric (Curcuma longa), without discernable toxicity, has been shown to inhibit the growth of colon tumor cells in vitro and in vivo partly by suppressing activation of NF-κB. The aim of this study was to demonstrate whether a combined treatment with CURC and OXA could revert the acquired resistance phenotype to the latter in in vitro models and to elucidate the molecular mechanisms behind this effect. Material and Methods: HT29- and LoVo-derived OXA-resistant HTOXAR3 and LoVOXAR3 cells (p53 mutant and WT respectively) as well as HCT116 and its isogenic derivative with a targeted inactivation of p53 (HCT116 p53 null) cell lines were used in the present study. The IC50s and combination index for CURC and OXA in the six cell lines was determined by MTT assay and the data was analysed using the median effect lines and the Chou and Talalay method, respectively. Survival and clonogenic expansion of cells after OXA and CURC treatment was assessed by colony assay. Cell death was measured by flow cytometry and propidium iodide staining. Dose-response-associated phosphorylation status of P65 (S536), IκBα(S32/S36) and expression of Survivin and CDK5 was analysed by Western Blotting after oxaliplatin and CURC treatments. Nuclear translocation of p65 was assessed by inmunocytochemistry. Results: CURC had a similar effect on proliferation in all cell lines (IC50 range 7.5-14.7uM). In terms of synergism, the best combination of CURC and OXA was the 24h concomitant treatment. CURC inhibited OXA-induced activation of NF-κB, downregulated the expression of Survivin, an NF-κB-regulated gene product, and it also inhibited CDK5 expression in a dose-dependent manner. Combination of OXA and CURC resulted in an OXA resistance reversion in HTOXAR3, LoVOXAR3 and HCT116 p53 null cells but it was only synergistic in HTOXAR3 and HCT116 p53 null cells, suggesting a possible role of p53 on OXA/CURC synergism. The concomitant treatment also reduced the number of colonies formed compared with OXA or CURC alone. This was further supported by preliminary experiments showing that the combination also increased cell death. Conclusions: These results demonstrate that the OXA/CURC combination can revert OXA acquired resistance in our resistant models through the inhibition of the NFκB TF and for the first time, suggest a role of CDK5 in the CURC mechanism of action. Further experiments are ongoing in order to elucidate a possible link between CDK5 and NF-κB, and their role in acquisition of OXA resistance. Citation Format: Vicenc Ruiz de Porras, Sara Bystrup, Anna Martinez-Cardus, Alba Gines, Laura Layos, Jose Luis Manzano, Cristina Buges, Albert Abad, Eva Martinez-Balibrea. Curcumin mediates reversion to oxaliplatin-acquired resistance in colorectal cancer cell lines through modulation of nuclear factor κB (NFκB) and cyclin-dependent kinase 5 (CDK5). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5478. doi:10.1158/1538-7445.AM2015-5478

Abstract 2610: TYMS and XRCC1 gene polymorphisms as possible predictors for the addition of oxaliplatin to 5FU in the adjuvant treatment of stage II and III colorectal cancer

Background: The MOSAIC trial demonstrated that adding oxaliplatin to LV5FU2 significantly improved 5-year disease free survival (DFS) and 6-year overall survival (OS) in the adjuvant treatment of stage II/III colorectal cancer (CRC). However, oxaliplatin-related toxicities represent a problem when managing these patients. Genetic variants in TYMS and XRCC1 have shown to influence patients9 outcomes in the metastatic setting. The purpose of the present study was to evaluate the influence of these polymorphisms on DFS and OS of stage II/III CRC patients treated with LV5FU2 +/- oxaliplatin in an adjuvant setting. Methods: We genotyped 216 patients (53.7% LV5FU2 and 46.3% FOLFOX4) from the MOSAIC trial. TYMS polymorphisms (5′TRP and 5′UTR SNP) and XRCC1-Arg399Gln were analyzed through PCR, RFLP and allelic discrimination respectively. According to TYMS 5′UTR SNP, genotypes were grouped as low expression (2R/2R, 2R/3RC, 3RC/3RC) or high expression (2R/3RG, 3RC/3RG, 3RG/3RG). Association of genetic variants with DFS and OS was investigated through Kaplan Meyer curves, log rank test and Cox regression models. Results: In our cohort 57.5% of patients were males; median age was 61 years (33-79); stage II/III tumors were equally distributed (50.2% and 49.8%). After 6 years of follow-up, 20.4% (38.6% FOLFOX4, 61.4% LV5FU2) of patients had relapsed and 15.4% (42.4% FOLFOX4, 57.6% LV5FU2) were dead. There were no statistical significant differences in DFS or OS according to individual genetic variants either in the whole group or by treatment. Therefore, they were analyzed jointly. TYMS high expression genotypes were considered as non-favorable for 5FU and XRCC1-399 Gln/Gln or Arg/Gln genotypes were considered as non-favorable for oxaliplatin. Patients in the FOLFOX4 group with non-favorable genotypes were more likely to relapse (28% vs. 11% for some favorable genotype; HR=2.7, 95% CI 1.00-7.34; Log rank p=0.04) or dying (17% vs. 5% for some favorable genotype; HR=2.7, 95% CI 0.92-8.17; Log rank p=0.06). Interestingly, these phenomena didn9t occur in the LV5FU2 group of patients. Taken together, patients with some favorable genotype had better outcomes when receiving FOLFOX4 (87% vs. 78% for DFS, HR=0.63, 95% CI 0.28-1.43; p=0.07 and 95% vs. 89% for OS, HR=0.56, 95% CI 0.27-1.15; p=ns) after 6 years of follow-up. Inversely, FOLFOX4 was detrimental for those patients with non-favorable genotypes as compared with patients receiving LV5FU2 (68% vs. 78% for DFS, HR=2.5, 95% CI 0.60-11.1; p=ns and 83% vs. 89% for OS, HR=1.27, 95% CI 0.41-4; p=ns). Conclusion: TYMS 5′UTR SNP and XRCC1 Arg399Gln genotyping could help in the decision of adding or not oxaliplatin to LV5FU2 as adjuvant treatment in patients with stage II/III CRC and thus avoid undesirable oxaliplatin-related toxicities to these patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2610.

Abstract 3752: PKM2 subcellular localization is involved in oxaliplatin resistance acquisition in human colorectal cancer cell lines

Background/purpose: Despite remaining one of the most active drugs in the treatment of advanced colorectal cancer (aCRC), oxaliplatin (OXA) resistance still is a multifactorial and complex process to decipher. In a previous work we identified low levels of PKM2 (mRNA and protein) as a putative OXA-resistance marker in CRC cell lines and patients. The purpose of this work was to identify molecular mechanisms underlying PKM2 role in resistance to OXA. Material and methods: PKM2 transient gene silencing was achieved by transfecting specific siRNAs (Ambion) in HT29 and HCT116 CRC cell lines. Gene silencing was validated by qPCR and Western Blot with specific Taqman primers and probes (assay no. Hs00762869_s1) and antibody (Cell signaling), respectively. Cell proliferation was measured by the MTT assay (Roche Diagnostics). Inhibitory concentrations were determined in each cell line by the median-effect line method. Cell viability was studied with the use of trypan blue stain and apoptosis was detected with FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen). PKM2 subcellular localization was studied by fluorescence microscopy in an Axiovision Z1 by using Apotome system at 40x immersion oil lens (Carl Zeiss, Heidelberg, Germany). Expression patterns of cell death genes were analyzed through Human Cell Death Pathway Finder PCR Array 384 HT (PAHS-212Z, SA Biosciences). Results: In order to assess how PKM2 expression influences OXA response in CRC cells, we silenced PKM2 by using specific siRNAs in HT29 and HCT116 cells. Cells were then treated with OXA at different concentrations and we observed that PKM2 silencing induced resistance in HT29 (p53 mutated) cells and sensitivity in HCT116 (p53 wild type) cells. Moreover, PKM2 knockdown was associated with an increase in cell viability but not with a decrease in apoptosis activation in HT29 cells. Fluorescence microscopy revealed PKM2 nuclear translocation in response to OXA in HCT116 and HT29 cells but not in OXA-resistant HTOXAR3 cells. Finally, by using a qPCR Array we demonstrated that OXA and PKM2 silencing altered cell death gene expression patterns including BMF, which was significantly increased in HT29 cells in response to OXA in a dose and time-dependent manner but not in siPKM2-HT29 and HTOXAR3 cells. Conclusion: Our data report new non-glycolytic roles of PKM2 in response to genotoxic damage, which depend on p53 mutational status and proposes BMF as a possible target gene of PKM2 to be involved in OXA response and resistance in CRC cells. Citation Format: Alba Gines, Anna Martinez-Cardus, Vicenc Ruiz de Porras, Eva Musulen, Jose Luis Manzano, Laura Layos, Cristina Buges, Albert Abad, Eva Martinez-Balibrea. PKM2 subcellular localization is involved in oxaliplatin resistance acquisition in human colorectal cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3752. doi:10.1158/1538-7445.AM2014-3752

Abstract 2546: IGR-1R pathway role in oxaliplatin acquired resistance in colon cancer cell lines and its regulation by MMP-7 and IGFBP-2

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: IGF-1R pathway and MMPs have been implicated in chemotherapy resistance in colorectal cancer. This receptor could be activated by IGF1 or IGF2 ligands, which bioavailability is regulated by IGFBPs, what could be degraded by MMPs. Objectives: To determine the basal levels and the modulation by chemotherapy (oxaliplatin), of the levels of IGF-1R, IGFBPs and MMPs in colon cancer cell lines (HT-29 (IC50: 6,46 uM) and LoVo (IC50: 0,25 uM) and their oxaliplatin resistant cell lines (HT-29OXAR3 (IC50: 29,86 uM) and LoVOXAR3 (2,45 uM)). To analyze the differences in apoptosis between parental and oxaliplatin resistant cell lines when are treated with oxaliplatin. Methods: MMP-7 levels were analyzed by RT-PCR, Western Blot and ELISA. Levels of IGF-1R, activated IGF-1R, IGFBP-2, and IGFBP-3 were determined by Western Blot and/or ELISA. Differences in cell viability were analyzed by MTT, and intrinsic (bcl-2, bax) and extrinsic (Fas, caspase 8) apoptotic pathways, by Western Blot, in parental and oxaliplatin resistant cell lines with dose-response experiments. Results: LoVo and LoVOXAR3 express low levels of MMP-7 and IGF-1R and high levels of IGFBP-2 compared with HT-29 and HT-29OXAR3. In addition, HT-29OXAR3 cells showed increased MMP-7, IGF-1R and pIGF-1R levels compared with HT-29 and lower levels of IGFBP-2. We also observed after oxaliplatin treatment in HT-29 cells a decrement of IGF-1R and IGFBP-2 and an increment in MMP-7 levels. In apoptotic experiments, FAS and caspase 8 (extrinsic pathway) were induced by oxaliplatin in LOVO (0.2 uM) and in LOVOXAR3 (20uM). Bcl-2 and bax (intrinsic pathway) were induced or decremented respectively in LOVO (2uM) and LOVOXAR3 (20uM). No changes in these extrinsic apoptotic proteins were observed in HT29 and HT29OXR3. In HT-29OXAR3 cell line higher doses of oxaliplatin (20 uM instead of 2uM) were needed to induce intrinsic apoptotic (bax) and antiapoptotic proteins (bcl-2) Conclusions: IGF-1R signalling is implicated in acquired oxaliplatin resistance and it is regulated by MMP-7 and IGFBP-2 in a different manner in our two models of cancer colon cell lines. The two apoptotic pathways were implicated in LoVo cell lines, but only intrinsic pathway regulates apoptosis in HT-29 cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2546.

Abstract 2562: Role of Pyruvate Kinase isozyme type M2 in response and resistance to oxaliplatin in HT29 human colon adenocarcinoma cell lines

BACKGROUND: Resistance to platinum-based chemotherapy is a multifactorial and complex process resulting in a major obstacle for effective treatment. In a previous work we identified low levels of PKM2 (mRNA and protein) as a putative oxaliplatin-resistance marker by using an in vitro model of resistance and a comparative proteomic approach. Our results were also validated in advanced colorectal cancer patients treated with oxaliplatin (Martinez-Balibrea, Molecular cancer therapeutics, 2009). PKM2 has been reported to translocate to the nucleus and activate apoptosis after H2O2, UV or a somatostatin analog treatment. The aim of this work was to study the role of PKM2 in the molecular mechanisms involved in response and resistance to oxaliplatin and identify proteins related to PKM2 expression levels. EXPERIMENTAL DESIGN: We silenced PKM2 expression by using siRNA techniques in the human adenocarcinoma cell line HT29. Validation of knockdown was done by Western Blotting (WB) and RTQPCR. To study the effect of PKM2 silencing in the resistance phenotype an MTT assay was performed to compare the viability between PKM2-silenced cells and those transfected with an unspecific siRNA (NTC). Cells were treated 24 hours with oxaliplatin at 15 µM. To verify the PKM2 nuclear translocation and its possible role in activating apoptosis after H2O2 and oxaliplatin treatment, HT29 and HCT116 cells were treated with H2O2 and oxaliplatin at equivalent doses and harvested at 0, 2 and 4h after treatment. Sub-cellular fractionation and WB as well as immunoflorescenece techniques were performed to visualize nuclear translocation of PKM2. To study possible changes in the proteome of HT29 cells as a consequence of PKM2 silencing, 2D- DIGE and Mass Spectrometry techniques were performed. RESULTS: Forty-eight hours after transfection we obtained a 95% of PKM2 knockdown by RTQPCR (mRNA level) and also by WB (protein level). A slightly increase of viability was observed in PKM2-silenced cells in comparison to NTC cells after oxaliplatin exposition (50% vs. 35%). Preliminary results showed that both oxaliplatin and H2O2 induced PKM2 nuclear translocation in HT29 and HCT116 cells 4 hours after the exposition to these agents. By 2D-DIGE we determined changes in the proteome of HT29 cells as a consequence of PKM2 silencing. The identification and validation of those proteins showing up- or down-regulation are in process and will be presented during the annual meeting. CONCLUSIONS: PKM2 knockdown alters sensitivity to oxaliplatin and protein expression patterns in HT29 cells. Oxaliplatin induces nuclear translocation of PKM2. Further studies are guaranteed to evaluate the role of PKM2 in activating apoptosis after oxaliplatin exposure. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2562.