Drug Resistance In Colorectal Cancer Research Articles

Positive feedback between oncogenic KRAS and HIF-1α confers drug resistance in colorectal cancer.

Approximately 30%–50% of colorectal cancers (CRCs) harbor the somatic mutated KRAS gene. KRAS G12V, one of the most common KRAS mutations in CRCs, is linked to increased tumor aggressiveness, less response to anti-epidermal growth factor receptor (EGFR) therapy, and poor survival rate. In this study, we sought to determine whether resistance to EGFR inhibitors in colorectal cancer cells harboring KRAS G12V mutation is associated with hypoxia. Our data indicated that HIF-1α was induced by KRAS G12V signaling at transcription level. Hypoxia or HIF-1α overexpression could increase KRAS G12V activity. Therefore, a positive feedback between hypoxia and KRAS G12V activation was formed. Cetuximab, an EGFR inhibitor, which has a minor effect on KRAS-mutant CRCs, could effectively inhibit the proliferation of CRC cells harboring KRAS G12V mutation when combined with HIF-1α inhibitor PX-478. Our data indicated that hypoxia was involved in resistance to anti-EGFR therapy, and a combination therapy might be necessary for CRC patients with KRAS mutation.

Identification of the sAPRIL binding peptide and its growth inhibition effects in the colorectal cancer cells.

BackgroundA proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) super family. It binds to its specific receptors and is involved in multiple processes during tumorigenesis and tumor cells proliferation. High levels of APRIL expression are closely correlated to the growth, metastasis, and 5-FU drug resistance of colorectal cancer. The aim of this study was to identify a specific APRIL binding peptide (BP) able to block APRIL activity that could be used as a potential treatment for colorectal cancer.MethodsA phage display library was used to identify peptides that bound selectively to soluble recombinant human APRIL (sAPRIL). The peptides with the highest binding affinity for sAPRIL were identified using ELISA. The effects of sAPRIL-BP on cell proliferation and cell cycle/apoptosis in vitro were evaluated using the CCK-8 assay and flow cytometry, respectively. An in vivo mouse model of colorectal cancer was used to determine the anti-tumor efficacy of the sAPRIL-BP.ResultsThree candidate peptides were characterized from eight phage clones with high binding affinity for sAPRIL. The peptide with the highest affinity was selected for further characterization. The identified sAPRIL-BP suppressed tumor cell proliferation and cell cycle progression in LOVO cells in a dose-dependent manner. In vivo in a mouse colorectal challenge model, the sAPRIL-BP reduced the growth of tumor xenografts in nude mice by inhibiting proliferation and inducing apoptosis intratumorally. Moreover, in an in vivo metastasis model, sAPRIL-BP reduced liver metastasis of colorectal cancer cells.ConclusionssAPRIL-BP significantly suppressed tumor growth in vitro and in vivo and might be a candidate for treating colorectal cancers that express high levels of APRIL.

The effectiveness of an anti-human IL-6 receptor monoclonal antibody combined with chemotherapy to target colon cancer stem-like cells.

Recent studies have demonstrated that cancer stem cells (CSCs) can initiate and sustain tumor growth and exhibit resistance to clinical cytotoxic therapies. Therefore, CSCs represent the main target of anticancer therapy. Interleukin-6 (IL-6) promotes cellular proliferation and drug resistance in colorectal cancer, and its serum levels correlate with patient survival. Therefore, IL-6 and its downstream signaling molecule the signal transducer and activator of transcription-3 (STAT3) represent potential molecular targets. In the present study, we investigated the effects of IL-6 and its downstream signaling components on stem cell biology, particularly the chemoresistance of CSCs, to explore potential molecular targets for cancer therapy. The colon cancer cell line WiDr was cultured in serum-free, non-adherent, and three-dimensional spheroid-forming conditions to enrich the stem cell-like population. Spheroid-forming cells slowly proliferated and expressed high levels of Oct-4, Klf4, Bmi-1, Lgr5, IL-6, and Notch 3 compared with adherent cells. Treatment with an anti-human IL-6 receptor monoclonal antibody reduced spheroid formation, stem cell-related gene expression, and 5-fluorouracil (5-FU) resistance. In addition, IL-6 treatment enhanced the levels of p-STAT3 (Tyr705), the expression of Oct-4, Klf4, Lgr5, and Notch 3, and chemoresistance to 5-FU. siRNA targeting Notch 3 suppressed spheroid formation, Oct-4 and Lgr5 expression, and 5-FU chemoresistance, whereas STAT3 inhibition enhanced Oct-4, Klf4, Lgr5, and Notch 3 expression and 5-FU chemoresistance along with reduced spheroid growth. Taken together, these results indicate that IL-6 functions in dichotomous pathways involving Notch 3 induction and STAT3 activation. The former pathway is involved in cancer stem-like cell biology and enhanced chemoresistance, and the latter pathway leads to accelerated proliferation and reduced chemoresistance. Thus, an anti-human IL-6 receptor monoclonal antibody or Notch 3 inhibition may be superior to STAT3 inhibition for CSC-targeting therapies concomitant with anticancer drugs.

Abstract CT320: Longitudinal observational study to simultaneously evaluate multiple blood-based biomarkers to track the emergence of metastasis and drug resistance in colorectal cancer

Abstract Background. Colorectal cancer (CRC) is the 3rd most common cancer globally. In cancer, the 2 most critical events that adversely affect outcomes are the emergence of metastasis and drug resistance. Blood-based markers permit the non-invasive serial monitoring of patients in order to study and track the emergence and evolution of metastasis and drug resistance. The multiple varied platforms utilize different components of blood (buffy coat, whole blood, plasma). We seek to establish a longitudinal observational study to simultaneously evaluate multiple blood-based biomarkers to track patient outcomes in colorectal cancer. Methods. In a planned 800 patient study, we prospectively recruit patients with all stages of CRC, focusing on patients with stage 3/4 CRC. At each patient's venipuncture performed during his/her routine clinical care, we serially collect additional blood specimens. All major components of the collected blood (buffy coat, red cells, whole blood and plasma) are stored. In patients undergoing primary tumor resection or metastasectomy, frozen tissue specimens are also prospectively collected and banked for subsequent biomarker analysis. A prospective electronic database of clinical information and patient outcomes is simultaneously maintained for the purpose of correlation with molecular assays. Results. To date, we have recruited 450 patients and obtained their serial blood samples. Blood is prospectively collected in an optimized manner to support the pre-analytical requirements of the different platforms. This includes whole blood for immediate assay performance (circulating tumor cells), buffy coat for immediate analysis or storage for pooled analysis (cell based assays, germline polymorphisms, white cell gene expression and assays on peripheral blood mononuclear cells), plasma for storage for pooled analysis (circulating cell free nucleic acids including DNA, miRNA, RNA and methylated DNA, novel proteomic tumor markers, cytokines). Optimization of pre-analytical processing, transport and storage parameters was carried out to enable studies across multiple platforms in this same cohort of patients to be performed in a prospective longitudinal manner. Conclusions. In this ongoing study, we have established a system for the simultaneous evaluation of multiple blood-based biomarkers operating on the various components of blood. Together with paired tissue specimens and a prospectively maintained electronic clinical database, comprehensive comparisons and correlative studies to monitor for metastasis and drug resistance and clinical outcomes in CRC across time can be carried out. Citation Format: Clarinda Chua, Rachel Ten, Thinzar Aung, Maricel Ang, Thein Htut Oo, Su Pin Choo, Matthew Ng, Iain Beehuat Tan. Longitudinal observational study to simultaneously evaluate multiple blood-based biomarkers to track the emergence of metastasis and drug resistance in colorectal cancer. [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 CT320. doi:10.1158/1538-7445.AM2014-CT320

Atmospheric pressure gas plasma-induced colorectal cancer cell death is mediated by Nox2–ASK1 apoptosis pathways and oxidative stress is mitigated by Srx–Nrf2 anti-oxidant system

Atmospheric pressure gas plasma (AGP) generates reactive oxygen species (ROS) that induce apoptosis in cultured cancer cells. The majority of cancer cells develop a ROS-scavenging anti-oxidant system regulated by Nrf2, which confers resistance to ROS-mediated cancer cell death. Generation of ROS is involved in the AGP-induced cancer cell death of several colorectal cancer cells (Caco2, HCT116 and SW480) by activation of ASK1-mediated apoptosis signaling pathway without affecting control cells (human colonic sub-epithelial myofibroblasts; CO18, human fetal lung fibroblast; MRC5 and fetal human colon; FHC). However, the identity of an oxidase participating in AGP-induced cancer cell death is unknown. Here, we report that AGP up-regulates the expression of Nox2 (NADPH oxidase) to produce ROS. RNA interference designed to target Nox2 effectively inhibits the AGP-induced ROS production and cancer cell death. In some cases both colorectal cancer HT29 and control cells showed resistance to AGP treatment. Compared to AGP-sensitive Caco2 cells, HT29 cells show a higher basal level of the anti-oxidant system transcriptional regulator Nrf2 and its target protein sulfiredoxin (Srx) which are involved in cellular redox homeostasis. Silencing of both Nrf2 and Srx sensitized HT29 cells, leads to ROS overproduction and decreased cell viability. This indicates that in HT29 cells, Nrf2/Srx axis is a protective factor against AGP-induced oxidative stress. The inhibition of Nrf2/Srx signaling should be considered as a central target in drug-resistant colorectal cancer treatments.

Bone morphogenetic protein 2 inhibits the proliferation and growth of human colorectal cancer cells.

Colorectal cancer (CRC) is one of the most deadly cancers worldwide. Significant progress has been made in understanding the molecular pathogenesis of CRC, which has led to successful early diagnosis, surgical intervention and combination chemotherapy. However, limited therapeutic options are available for metastatic and/or drug-resistant CRC. While the aberrantly activated Wnt/β-catenin pathway plays a critical initiating role in CRC development, disruption of the bone morphogenetic protein (BMP) pathway causes juvenile polyposis syndrome, suggesting that BMP signaling may play a role in CRC development. However, conflicting results have been reported concerning the possible roles of BMP signaling in sporadic colon cancer. Here, we investigated the effect of BMP2 on the proliferation, migration, invasiveness and tumor growth capability of human CRC cells. Using an adenovirus vector that overexpresses BMP2 and the piggyBac transposon-mediated stable BMP2 overexpression CRC line, we found that exogenous BMP2 effectively inhibited HCT116 cell proliferation and colony formation. BMP2 was shown to suppress colon cancer cell migration and invasiveness. Under a low serum culture condition, forced expression of BMP2 induced a significantly increased level of apoptosis in HCT116 cells. Using a xenograft tumor model, we found that forced expression of BMP2 in HCT116 cells suppressed tumor growth, accompanied by decreased cell proliferation activity. Taken together, our results strongly suggest that BMP2 plays an important inhibitory role in governing the proliferation and aggressive features of human CRC cells.

Fibroblast growth factor receptor 4 (FGFR4): a targetable regulator of drug resistance in colorectal cancer

The discovery of underlying mechanisms of drug resistance, and the development of novel agents to target these pathways, is a priority for patients with advanced colorectal cancer (CRC). We previously undertook a systems biology approach to design a functional genomic screen and identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of drug resistance. The aim of this study was to examine the role of FGFR4 in drug resistance using RNAi and the small-molecule inhibitor BGJ398 (Novartis). We found that FGFR4 is highly expressed at the RNA and protein levels in colon cancer tumour tissue compared with normal colonic mucosa and other tumours. Silencing of FGFR4 reduced cell viability in a panel of colon cancer cell lines and increased caspase-dependent apoptosis. A synergistic interaction was also observed between FGFR4 silencing and 5-fluorouracil (5-FU) and oxaliplatin chemotherapy in colon cancer cell lines. Mechanistically, FGFR4 silencing decreased activity of the pro-survival STAT3 transcription factor and expression of the anti-apoptotic protein c-FLIP. Furthermore, silencing of STAT3 resulted in downregulation of c-FLIP protein expression, suggesting that FGFR4 may regulate c-FLIP expression via STAT3. A similar phenotype and downstream pathway changes were observed following FGFR4 silencing in cell lines resistant to 5-FU, oxaliplatin and SN38 and upon exposure of parental cells to the FGFR small-molecule inhibitor BGJ398. Our results indicate that FGFR4 is a targetable regulator of chemo-resistance in CRC, and hence inhibiting FGFR4 in combination with 5-FU and oxaliplatin is a potential therapeutic strategy for this disease.

Uptake of Etoposide in CT-26 Cells of Colorectal Cancer Using Folate Targeted Dextran Stearate Polymeric Micelles

Targeted drug delivery using folate receptors is one of the most interesting chemotherapeutic research areas over the past few years. A novel folate targeted copolymer was synthesized using dextran stearate coupled to folic acid. FT-IR and NMR spectroscopy were used to confirm successful conjugation. Micelles prepared using this copolymer were characterized for their particle size, zeta potential, critical micelle concentration (CMC), drug loading capacity, and release efficiency. Cytotoxicity and cellular uptake of the micelles were estimated using CT-26 colorectal carcinoma cell line. FT-IR and NMR spectroscopy confirmed production of folate grafted dextran stearate copolymer. Low CMC value indicates that the copolymers are suitable for preparation of stable micelles useful in parenteral dosage forms. Particle size and zeta potential of the targeted nanoparticles were 105.5 ± 2.0 nm and −21.2 mV, respectively. IC50 of etoposide loaded in folate grafted dextran stearate enhanced about 20-fold compared to the pure drug (0.49 ± 0.11 μg/mL versus 9.41 ± 0.52 μg/mL). It seems that etoposide loaded in micelles of folate grafted dextran stearate copolymer is promising in reducing drug resistance of colorectal cancer by boosting etoposide cellular uptake.

Neoamphimedine, a Lead Anti-Metastatic Agent for the Treatment of Drug Resistant and Metastatic Colorectal Cancer

Neoamphimedine, a Lead Anti-Metastatic Agent for the Treatment of Drug Resistant and Metastatic Colorectal Cancer

MicroRNAs in Colorectal Cancer Drug Resistance: Shooters become Targets

Copyright: © 2013 Fanale D, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. MicroRNAs (MiRNAs) are involved in the regulation of several biological processes such as development, differentiation, metabolism, apoptosis and proliferation. Recently, it has been shown that deregulated expression of miRNAs are present in different human cancers, suggesting a potential role in carcinogenesis [1,2]. Recent evidence suggests that miRNAs may represent potential new therapeutic approaches in patients with drug resistance and drug induced toxicity [3]. MiRNAs may have therapeutic applications through two mechanisms. The first strategy involves the inhibition of oncogenic miRNAs by using miRNA antagonists (anti-miRNAs) resulting in up-regulation of genes that would be silenced by deregulated miRNAs. The second strategy involves the restoration in expression levels of tumor suppressor microRNA with miRNA mimics [1]. To date, several studies have shown that miRNA mimics and antimiRNAs may be useful to restore normal gene networks in different cancer cell lines and animal models, suggesting a new potential role in anti-cancer therapy.

Abstract 908: Differential role of JAK1/2-STAT3 pathway in drug response and survival of oncogenic Kras colorectal cancer

Abstract Mutations in the Kras proto-oncogene are found in 40%-45% of colorectal cancer (CRC) patients and confer resistance to EGFR monoclonal antibody (mAb) therapies. The identification of druggable targets uniquely required by KrasMT CRC tumours has the potential to fill a gap in the therapeutic armamentarium of advanced CRC. We carried out microarray profiling of isogenic Kras wild type (WT) and mutant (MT) in vivo CRC models, KrasMT/WT primary colon tumours, pre-treatment metastatic CRC liver biopsies and drug resistant and sensitive CRC cell lines. We have used systems biology approach incorporating pathway analyses of the microarray data followed by siRNA screening to identify novel targets/pathways which are critical for the survival of KrasMT tumours, both basally and following chemotherapy treatment. We found that JAK1/2-STAT3 signaling was essential for the survival of KrasMT CRC cells. Moreover, suppression of JAK1/2-STAT3 activity using RNAi or small molecule inhibitors resulted in more potent increase in apoptosis in KrasMT CRC cells. In addition, treatment with chemotherapy and MEK1/2 inhibition resulted in significant increases in JAK2 and STAT3 activity and this was associated with strong increase in apoptosis when JAK1/2-STAT3 RNAi or small molecule inhibitors were combined with chemotherapy and MEK1/2 inhibition, especially in KrasMT cells. Moreover, we found that these effects were not drug- or cell line-specific. Collectively, using a systems biology approach, we have identified an essential role of JAK1/2-STAT3 signalling in survival of KrasMT CRC models. These findings support the further clinical investigation of JAK/STAT3 inhibitors in conjunction with chemotherapy or MEK inhibition in KrasMT CRC tumours. 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 908. doi:1538-7445.AM2012-908

A Systems Biology Approach Identifies SART1 as a Novel Determinant of Both 5-Fluorouracil and SN38 Drug Resistance in Colorectal Cancer

Chemotherapy response rates for advanced colorectal cancer remain disappointingly low, primarily because of drug resistance, so there is an urgent need to improve current treatment strategies. To identify novel determinants of resistance to the clinically relevant drugs 5-fluorouracil (5-FU) and SN38 (the active metabolite of irinotecan), transcriptional profiling experiments were carried out on pretreatment metastatic colorectal cancer biopsies and HCT116 parental and chemotherapy-resistant cell line models using a disease-specific DNA microarray. To enrich for potential chemoresistance-determining genes, an unsupervised bioinformatics approach was used, and 50 genes were selected and then functionally assessed using custom-designed short interfering RNA (siRNA) screens. In the primary siRNA screen, silencing of 21 genes sensitized HCT116 cells to either 5-FU or SN38 treatment. Three genes (RAPGEF2, PTRF, and SART1) were selected for further analysis in a panel of 5 colorectal cancer cell lines. Silencing SART1 sensitized all 5 cell lines to 5-FU treatment and 4/5 cell lines to SN38 treatment. However, silencing of RAPGEF2 or PTRF had no significant effect on 5-FU or SN38 sensitivity in the wider cell line panel. Further functional analysis of SART1 showed that its silencing induced apoptosis that was caspase-8 dependent. Furthermore, silencing of SART1 led to a downregulation of the caspase-8 inhibitor, c-FLIP, which we have previously shown is a key determinant of drug resistance in colorectal cancer. This study shows the power of systems biology approaches for identifying novel genes that regulate drug resistance and identifies SART1 as a previously unidentified regulator of c-FLIP and drug-induced activation of caspase-8.

MicroRNA-451 Is Involved in the Self-renewal, Tumorigenicity, and Chemoresistance of Colorectal Cancer Stem Cells

Many antitumor therapies affect rapidly dividing cells. However, tumor proliferation may be driven by cancer stem cells (CSCs), which divide slowly and are relatively resistant to cytotoxic drugs. Thus, many tumors may progress because CSCs are not sensitive to the treatment. In this work, we searched for target genes whose expression is involved in proliferation and chemoresistance of CSCs. Both of these processes could be controlled simultaneously by cell regulators such as microRNAs (miRNAs). Therefore, colonospheres with properties of CSCs were obtained from different colon carcinoma cells, and miRNA profiling was performed. The results showed that miR-451 was downregulated in colonspheres versus parental cells. Surprisingly, expression of miR-451 caused a decrease in self-renewal, tumorigenicity, and chemoresistance to irinotecan of colonspheres. We identified cyclooxygenase-2 (COX-2) as an indirect miR-451 target gene involved in sphere growth. Our results indicate that miR-451 downregulation allows the expression of the direct target gene macrophage migration inhibitory factor, involved in the expression of COX-2. In turn, COX-2 allows Wnt activation, which is essential for CSC growth. Furthermore, miR-451 restoration decreases expression of the ATP-binding cassette drug transporter ABCB1 and results in irinotecan sensitization. These findings correlate well with the lower expression of miR-451 observed in patients who did not respond to irinotecan-based first-line therapy compared with patients who did. Our data suggest that miR-451 is a novel candidate to circumvent recurrence and drug resistance in colorectal cancer and could be used as a marker to predict response to irinotecan in patients with colon carcinoma.

Epigenetics and chemoresistance in colorectal cancer: An opportunity for treatment tailoring and novel therapeutic strategies

Colorectal cancer is the second leading cause of cancer-related deaths in the world. Despite many therapeutic opportunities, prognosis remains dismal for patients with metastatic disease, and a significant portion of early-stage patients develop recurrence after chemotherapy. Epigenetic gene regulation is a major mechanism of cancer initiation and progression, through the inactivation of several tumor suppressor genes. Emerging evidence indicates that epigenetics may also play a key role in the development of chemoresistance. In the present review, we summarize epigenetic mechanisms triggering resistance to three commonly used agents in colorectal cancer: 5-fluorouracil, irinotecan and oxaliplatin. Those epigenetic biomarkers may help stratify colorectal cancer patients and develop a tailored therapeutic approach. In addition, epigenetic modifications are reversible through specific drugs: histone-deacetylase and DNA-methyl-transferase inhibitors. Preclinical studies suggest that these drugs may reverse chemoresistance in colorectal tumors. In conclusion, an epigenetic approach to colorectal cancer chemoresistance may pave the way to personalized treatment and to innovative therapeutic strategies.

Lymph Node Stromal Cells Enhance Drug-Resistant Colon Cancer Cell Tumor Formation through SDF-1α/CXCR4 Paracrine Signaling

Colorectal cancer (CRC) is the third most common malignancy and the second leading cause of cancer-related deaths in America. Nearly two thirds of newly diagnosed CRC cases include lymph node (LN) involvement, and LN metastasis is one of the strongest negative prognostic factors for CRC. It is thought that CRC tumors contain a small population of drug-resistant CRC tumor-initiating cells (Co-TICs) that may be responsible for cancer recurrence. To evaluate the effects of the LN stromal cells on Co-TICs, we established a unique xenoplant model using CRC cells isolated by enzymatic digestion from consented patient specimens, HT-29 cells, HCA-7 cells, and LN stromal cell line HK cells. We found that HK cells and HK cell-conditioned media enhanced CRC tumor formation and tumor angiogenesis. Cells expressing CD133+ and the stromal cell-derived factor 1α (SDF-1α) receptor CXCR4 were enriched in chemotherapeutic-resistant CRC cells. CD133+CXCR4+ Co-TICs isolated from patient specimens are more tumorigenic than unsorted tumor cells. Furthermore, the inhibitors specific to HK cell-derived SDF-1α reduced tumor formation and tumor angiogenesis. Our results have demonstrated a role for Co-TICs in tumor growth and defined the influence of LN stromal cells on Co-TICs. We have identified a major Co-TIC/LN microenvironment-specific mechanism for CRC resistance to chemotherapeutic agents and established experimental platforms for both in vitro and in vivo testing, indicating that SDF-1α and its receptor, CXCR4, may be targets for clinical therapy.

Abstract 4075: Chemoresistant colorectal cancer cells exhibit high glycolytic activity

Abstract Background: Overcoming drug resistance in colorectal cancer (CRC) requires an understanding the mechanisms by which cancer cells adapt to the genotoxic stress. We established oxaliplatin resistant CRC cells derived from HCT116 and HT29 cell lines (HCT116 OxR and HT29 OxR) (Yang AD et al, Clin Ca Res, 2007). The mechanisms by which these resistant clones reprogram their energy metabolism to gain a survival advantage remain unknown. In this study, we hypothesized that chemoresistant CRC cells would exhibit altered cellular metabolism in order to survive following chronic genotoxic stress. Methods: The expression level of the glycolytic enzymes Glut1, hexokinase II (HK2), LDHA and HIF1α were detected by Western Blotting. Glucose uptake and lactate production was calculated by measuring the concentration of glucose and lactate in the culture media. Intracellular ATP/ADP levels, oxygen consumption and mitochondria ATP production were quantified. An in vivo xenograft study was used to compare the growth rate and angiogenesis of parental and oxaliplatin-resistant cells. Results: Compared with the parental cells, both HT29-OxR and HCT116-OxR cells exhibited a metabolic phenotype with increased glycolysis as reflected by increased glucose uptake and lactate production. Glycolytic enzymes were upregulated in resistant cells including Glut1, HK2 and LDHA. HIF1α expression and VEGF levels in the conditioned media were increased in the resistant cells. The mitochondria of OxR cells demonstrated defective complex I/II substrate ATP production despite increased cellular oxygen consumption. Importantly, the OxR cells maintain higher levels of intracellular ATP and ATP/ADP ratio indicating a metabolic switch to glycolysis. In a tumor xenograft model, HT29 OxR cells grew significantly slower than parental HT29 cells. Interestingly, when OxR cells were mixed with parental HT29 cells (50:50 and 90:10), tumor growth and microvessel counts were significantly increased. Conclusion: Oxaliplatin-resistant cells demonstrated: 1) high aerobic glycolytic activity, 2) an increase in HIF1α and glycolytic enzymes, and 3) defective mitochondria function. This metabolic switch provides more ATP production and likely contributes to the chemoresistant phenotype. Altering energy metabolism may provide a novel strategy to overcome drug-resistance in CRC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4075. doi:10.1158/1538-7445.AM2011-4075

Evaluation of glycolytic activity and HIF-1α expression in chemoresistant colorectal cancer cells.

415 Background: Overcoming drug resistance in colorectal cancer (CRC) involves understanding the mechanisms by which cancer cells adapt to the genotoxic stress. We established a model of oxaliplatin resistant cells derived from HCT116/HT29 (HCT116 OxR/HT29 OxR). How these resistant clones reprogram their energy metabolism to gain a survival advantage remains unknown. We hypothesized that chemoresistant CRC cells exhibit altered cellular metabolism to survive following chronic genotoxic stress. Methods: The expression levels of glycolytic enzymes were detected by Western Blotting. Glucose and lactate concentrations were measured in the culturing media (CM). Intracellular ATP/ADP levels, oxygen consumption rate (OCR) and mitochondria ATP production were measured. In vivo xenograft study was used to compare the growth rate and angiogenesis of parental and oxaliplatin-resistant cells. Results: Both HT29-OxR and HCT116-OxR cells compared to parental cells exhibit increased aerobic glycolysis. Consistently, Glut1, Hexokinase 2 and LDHA glycolytic enzymes are upregulated. The OxR cells are defective in mitochondria complex I/II substrate and ATP production despite increased OCR. Importantly, the OXR cells maintain higher intracellular ATP and ATP/ADP ratio, increased HIF1a and VEGF levels in the CM. In tumor xenograft model, HT29 OxR cells grow significant slower than parental HT29 cells. Interestingly, when OxR cells were mixed with parental HT29 cells (50:50 and 90:10), tumor growth and microvessel counts were significantly accelerated. Conclusions: OxR cells demonstrate: 1) high aerobic glycolytic activity; 2) defective mitochondria functions; 3) increased ATP production; 4) increased HIF1a and angiogenesis. Likely, this metabolic switch contributes to the resistant phenotype. Metabolism intervention such as targeting HIF1a may provide a novel strategy to overcome drug-resistance in CRC. No significant financial relationships to disclose.

Chemotherapy-Induced Activation of ADAM-17: A Novel Mechanism of Drug Resistance in Colorectal Cancer

We have shown previously that exposure to anticancer drugs can trigger the activation of human epidermal receptor survival pathways in colorectal cancer (CRC). In this study, we examined the role of ADAMs (a disintegrin and metalloproteinases) and soluble growth factors in this acute drug resistance mechanism. In vitro and in vivo models of CRC were assessed. ADAM-17 activity was measured using a fluorometric assay. Ligand shedding was assessed by ELISA or Western blotting. Apoptosis was assessed by flow cytometry and Western blotting. Chemotherapy (5-fluorouracil) treatment resulted in acute increases in transforming growth factor-alpha, amphiregulin, and heregulin ligand shedding in vitro and in vivo that correlated with significantly increased ADAM-17 activity. Small interfering RNA-mediated silencing and pharmacologic inhibition confirmed that ADAM-17 was the principal ADAM involved in this prosurvival response. Furthermore, overexpression of ADAM-17 significantly decreased the effect of chemotherapy on tumor growth and apoptosis. Mechanistically, we found that ADAM-17 not only regulated phosphorylation of human epidermal receptors but also increased the activity of a number of other growth factor receptors, such as insulin-like growth factor-I receptor and vascular endothelial growth factor receptor. Chemotherapy acutely activates ADAM-17, which results in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. Thus, pharmacologic inhibition of ADAM-17 in conjunction with chemotherapy may have therapeutic potential for the treatment of CRC.

Using Pharmacogenomic Tumor Profiling to Identify Biomarkers of 5-fluorouracil Response in Colorectal Cancer

The chemotherapeutic agent 5-fluorouracil (5-FU) remains the drug of choice for the treatment of metastatic colorectal cancer, despite the fact that poor patient response to the drug due to acquired or inherent drug resistance is a major clinical problem. To be able to predict an individual patients response to 5-FU would facilitate the design of chemotherapeutic regimens tailored according to the individual patient and tumor profile. Pharmacogenomic studies provide information as to how variability on a genome-wide scale influences drug response, and are facilitated by high throughput technologies such as genomic and expression microarrays and single nucleotide polymorphism (SNP) assays. Not surprisingly, such investigations are taking the place of genetic studies that investigate associations between drug response and genetic alterations for one individual gene. This reflects the growing awareness that cancer drug response is multi-faceted. The cellular mechanisms underlying drug resistance can involve alterations at the single gene and/or genomic level on multiple biological regulatory pathways, e.g., cell cycle progression, apoptotic, and DNA repair pathways. This review will provide an update on how pharmacogenomic studies are being used to dissect the cellular mechanisms underlying drug response and drug resistance in colorectal cancer in order to identify novel biomarkers of 5-FU response, and how knowledge gained from transcriptional profiling studies of colorectal tumors is serving as a guide for optimization of current chemotherapeutic strategies and for design of new treatment strategies.

Clinical and pharmacologic study of multidrug resistance reversal with vinblastine and bepridil.

To achieve an adequate plasma concentration of bepridil, a calcium channel blocker, which reverts multidrug resistance (MDR) in vitro, when administered in combination with vinblastine in patients with advanced colorectal cancer, a tumor characterized by high MDR1 gene expression. To study the pharmacokinetics of both drugs, tolerability and antitumor activity in relation to the MDR1 expression in tumor tissue. Sixteen colorectal cancer patients entered the study. Bepridil was administered by central venous catheter as 5-mg/kg bolus over 30 minutes, followed by 12 mg/kg for 12 hours and 5 mg/kg for 24 hours. Vinblastine 5 mg/m2 was administered as an intravenous (i.v.) bolus 24.5 hours after the start of bepridil. MDR1/P-glycoprotein (Pgp) expression was assessed in 14 tumor samples by immunohistochemistry and RNase protection assay. The bepridil plasma level was greater than 2 mumol/L at the time of vinblastine administration in all patients investigated. At the dose used in the study, bepridil produced a QTc-prolongation more than 50 ms, which prevented further dose escalation. However, cardiac toxicity was asymptomatic in all treated patients, and other side effects were mild. MDR1/Pgp expression was positive in nine of 14 cases. Of fifteen patients assessable for response, one complete remission of 8 months' duration and 14 progressions were observed. The responding patient attained complete remission again when re-treated on progression with vinblastine alone. Bepridil plasma concentrations needed in vitro to modulate MDR could be achieved in this study with tolerable toxicity; however, despite most tumors being MDR1/Pgp-positive, no response was obtained that could be attributed to the drug combination. Mechanisms of drug resistance other than MDR are probably implicated in drug resistance of colorectal cancer.