Sensitivity Of Colorectal Cancer Cells Research Articles

Abstract 1474: Hepatocytes either radioprotect or sensitize colorectal cancer cells through nitric oxide-induced oxygen sparing in hypoxic microenvironment

Abstract Purpose: Impaired tumor oxygenation remains a major obstacle to curative radiotherapy in advanced primary tumors and metastases. In this study, tailored to the liver metastases of colorectal cancer (CRC), we compared the ability of tumor cells and normal host hepatocytes to metabolically consume oxygen and induce hypoxia. Next, we examined the possibility to re-oxygenate and thereby radiosensitize human CRC cells through the physiological mediator nitric oxide (NO) that switches the mitochondrial oxygen metabolism in hepatocytes from consumption to sparing. Methods and Materials: To model tumor-relevant hypoxia, hepatocytes and tumor cells were incubated in a tissue-mimetic co-culture system with limited oxygenation, which was monitored by fluorescence and linked to hypoxic radioresponse. To activate NO production through inducible nitric oxide synthase (iNOS), hepatocytes (or tumor cells) were exposed to a cytokine mixture of IFN-γ, TNF-α, IL-1α and LPS, and iNOS expression was examined by RT-PCR, western blotting, and NO/nitrite production. Results: Primary mouse hepatocytes consumed 10 to 40-times more oxygen than mouse CT26 and human DLD-1, HT29, HCT116 and SW480 CRC cells, suggesting that hepatocytes are the major effectors of hypoxic conditioning. At cell densities of 0.6-1.2 mln/ml, hepatocytes induced radiobiologically-relevant hypoxia within 20 min, and thereby radioprotected tumor cells 2.0 to 2.5-times. Following exposure to cytokines, hepatocytes revealed significant activation of iNOS-mediated production of NO that targeted mitochondrial aconitase and complexes II, and induced a profound respiratory self-arrest at up to 90 min. As a result, the spared oxygen caused uniform re-oxygenation and radiosensitization in a panel of CRC cell lines (by 1.9 to 2.7-times). Next, both oxygen sparing and radiosensitization could be counteracted by aminoguanidine, a metabolic iNOS inhibitor. Contrasting, all CRC cell lines showed little if any iNOS expression and therefore could not contribute to the oxygen-sparing and radiosensitizing effects of NO-producing hepatocytes. Finally, the NO donor S-nitroso-N-acetylpenicillamine and the mitochondrial inhibitors rotenone, antimycine A and sodium azide were able to block the mitochondrial respiratory chain and to reverse both metabolic hypoxia and hypoxia-induced radioresistance. Conclusion: Hepatocytes may profoundly modulate the hypoxic microenvironment and either protect or sensitize CRC cells to radiation, dependent on the mitochondrial respiratory switch. Our study for the first time provides evidence that host liver cells possess intrinsic radiosensitizing potential through NO-induced oxygen sparing. 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 1474. doi:1538-7445.AM2012-1474

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

Induced pluripotent stem cell-related genes influence biological behavior and 5-fluorouracil sensitivity of colorectal cancer cells

We aimed to perform a preliminary study of the association between induced pluripotent stem cell (iPS)-related genes and biological behavior of human colorectal cancer (CRC) cells, and the potential for developing anti-cancer drugs targeting these genes. We used real-time reverse transcriptase polymerase chain reaction (RT-PCR) to evaluate the transcript levels of iPS-related genes NANOG, OCT4, SOX2, C-MYC and KLF4 in CRC cell lines and cancer stem cells (CSCs)-enriched tumor spheres. NANOG was knockdowned in CRC cell line SW620 by lentiviral transduction. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, plate colony formation, and a mouse xenograft model were used to evaluate alterations in biological behavior in NANOG-knockdown SW620 cells. Also, mock-knockdown and NANOG-knockdown cells were treated with 5-fluorouracil (5-FU) and survival rate was measured by MTT assay to evaluate drug sensitivity. A significant difference in the transcript levels of iPS-related genes between tumor spheres and their parental bulky cells was observed. NANOG knockdown suppressed proliferation, colony formation, and in vivo tumorigenicity but increased the sensitivity to 5-FU of SW620 cells. 5-FU treatment greatly inhibited the expression of the major stemness-associated genes NANOG, OCT4, and SOX2. These results collectively suggest an overlap between iPS-related genes and CSCs in CRC. Quenching a certain gene NANOG may truncate the aggressiveness of CRC cells.

Sorcin Induces a Drug-Resistant Phenotype in Human Colorectal Cancer by Modulating Ca2+ Homeostasis

The Ca(2+)-binding protein sorcin regulates intracellular calcium homeostasis and plays a role in the induction of drug resistance in human cancers. Recently, an 18 kDa mitochondrial isoform of sorcin was reported to participate in antiapoptosis in human colorectal cancer (CRC), but information remains lacking about the functional role of the more abundant 22 kDa isoform of sorcin expressed in CRC. We found the 22 kDa isoform to be widely expressed in human CRC cells, whether or not they were drug resistant. Its upregulation in drug-sensitive cells induced resistance to 5-fluorouracil, oxaliplatin, and irinotecan, whereas its downregulation sensitized CRC cells to these chemotherapeutic agents. Sorcin enhances the accumulation of Ca(2+) in the endoplasmic reticulum (ER), preventing ER stress, and, in support of this function, we found that the 22 kDa isoform of sorcin was upregulated under conditions of ER stress. In contrast, RNAi-mediated silencing of sorcin activated caspase-3, caspase-12, and GRP78/BiP, triggering apoptosis through the mitochondrial pathway. Our findings establish that CRC cells overexpress sorcin as an adaptive mechanism to prevent ER stress and escape apoptosis triggered by chemotherapeutic agents, prompting its further investigation as a novel molecular target to overcome MDR.

S100A10 protein expression is associated with oxaliplatin sensitivity in human colorectal cancer cells

BackgroundIndividual responses to oxaliplatin (L-OHP)-based chemotherapy remain unpredictable. The objective of our study was to find candidate protein markers for tumor sensitivity to L-OHP from intracellular proteins of human colorectal cancer (CRC) cell lines. We performed expression difference mapping (EDM) analysis of whole cell lysates from 11 human CRC cell lines with different sensitivities to L-OHP by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), and identified a candidate protein by liquid chromatography/mass spectrometry ion trap time-of-flight (LCMS-IT-TOF).ResultsOf the qualified mass peaks obtained by EDM analysis, 41 proteins were differentially expressed in 11 human colorectal cancer cell lines. Among these proteins, the peak intensity of 11.1 kDa protein was strongly correlated with the L-OHP sensitivity (50% inhibitory concentrations) (P < 0.001, R2 = 0.80). We identified this protein as Protein S100-A10 (S100A10) by MS/MS ion search using LCMS-IT-TOF. We verified its differential expression and the correlation between S100A10 protein expression levels in drug-untreated CRC cells and their L-OHP sensitivities by Western blot analyses. In addition, S100A10 protein expression levels were not correlated with sensitivity to 5-fluorouracil, suggesting that S100A10 is more specific to L-OHP than to 5-fluorouracil in CRC cells. S100A10 was detected in cell culture supernatant, suggesting secretion out of cells.ConclusionsBy proteomic approaches including SELDI technology, we have demonstrated that intracellular S100A10 protein expression levels in drug-untreated CRC cells differ according to cell lines and are significantly correlated with sensitivity of CRC cells to L-OHP exposure. Our findings provide a new clue to searching predictive markers of the response to L-OHP, suggesting that S100A10 is expected to be one of the candidate protein markers.

Methyl jasmonate down-regulates survivin expression and sensitizes colon carcinoma cells towards TRAIL-induced cytotoxicity

Methyl jasmonate (MJ) is a plant stress hormone with selective cytotoxic anti-cancer activities. The TNF-related apoptosis-inducing ligand (TRAIL) death pathway is an attractive target for cancer therapy. Although TRAIL receptors are specifically expressed in primary cancer cells and cancer cell lines, many types of cancer cells remain resistant to TRAIL-induced cytotoxicity. Here we have assessed a possible synergy between MJ and TRAIL cytotoxicity in colorectal cancer (CRC) cell lines. CRC cell lines were pre-incubated with sub-cytotoxic concentrations of MJ followed by TRAIL administration. Cell death was determined by XTT assay and microscopy. Cytochrome c release, caspase cleavage, TRAIL-associated factors, X-linked inhibitor of apoptosis (XIAP) and survivin protein levels were detected by immunoblotting. Survivin transcription was examined by RT-PCR. Pre-treatment with MJ resulted in increased TRAIL-induced apoptotic cell death, increased cytochrome c release and caspase cleavage. TNFRSF10A, TNFRSF10B, TNFRSF10D, Fas-associated death domain and cellular FLICE-like inhibitory protein remained unchanged during MJ-induced TRAIL sensitization, whereas MJ induced a significant decrease in survivin protein levels. Overexpression of survivin prevented MJ-induced TRAIL cytotoxicity, implying a role for survivin in MJ-induced TRAIL sensitization. MJ decreased survivin mRNA indicating that MJ may affect survivin transcription. In a β-catenin/transcription factor (TCF)-dependent luciferase activity assay, MJ decreased TCF-dependent transcriptional activity. MJ, at sub-cytotoxic levels, sensitized CRC cells to TRAIL-induced apoptosis. Thus, combinations of MJ and TRAIL, both selective anti-cancer agents, have potential as novel treatments for CRC.

Silencing of the Wnt transcription factor TCF4 sensitizes colorectal cancer cells to (chemo-) radiotherapy

A considerable percentage of rectal cancers are resistant to standard preoperative chemoradiotherapy. Because patients with a priori-resistant tumors do not benefit from multimodal treatment, understanding and overcoming this resistance remains of utmost clinical importance. We recently reported overexpression of the Wnt transcription factor TCF4, also known as TCF7L2, in rectal cancers that were resistant to 5-fluorouracil-based chemoradiotherapy. Because Wnt signaling has not been associated with treatment response, we aimed to investigate whether TCF4 mediates chemoradioresistance. RNA interference-mediated silencing of TCF4 was employed in three colorectal cancer (CRC) cell lines, and sensitivity to (chemo-) radiotherapy was assessed using a standard colony formation assay. Silencing of TCF4 caused a significant sensitization of CRC cells to clinically relevant doses of X-rays. This effect was restricted to tumor cells with high T cell factor (TCF) reporter activity, presumably in a β-catenin-independent manner. Radiosensitization was the consequence of (i) a transcriptional deregulation of Wnt/TCF4 target genes, (ii) a silencing-induced G(2)/M phase arrest, (iii) an impaired ability to adequately halt cell cycle progression after radiation and (iv) a compromised DNA double strand break repair as assessed by γH2AX staining. Taken together, our results indicate a novel mechanism through which the Wnt transcription factor TCF4 mediates chemoradioresistance. Moreover, they suggest that TCF4 is a promising molecular target to sensitize resistant tumor cells to (chemo-) radiotherapy.

Abstract 4196: MSH3 mediates sensitization of colorectal cancer cells to cisplatin, oxaliplatin and a poly(ADP-ribose) polymerase inhibitor

Abstract Introduction:The MSH3 gene is one of the DNA mismatch repair (MMR) genes that has undergone somatic mutation frequently in MMR-deficient cancers. MSH3, together with MSH2 forms the MutSβ heteroduplex, which interacts with interstrand crosslinks (ICLs) induced by drugs such as cisplatin and psoralen. However, the precise role of MSH3 in mediating the cytotoxic effects of ICL-inducing agents remains poorly understood. MethodsIn this study, we first examined the effects of MSH3 deficiency on cytotoxicity caused by cisplatin and oxaliplatin, another ICL-inducing platinum drug. Results: Using isogenic HCT116-derived clones in which MSH3 expression is controlled by shRNA expression in a tet-off system, we discovered that MSH3 deficiency sensitized cells to both cisplatin and oxaliplatin at clinically relevant doses. Interestingly, siRNA-induced down-regulation of the MLH1 protein did not affect MSH3-dependent toxicity of these drugs, indicating that this process does not require participation of the canonical MMR pathway. Furthermore, MSH3-deficient cells maintained higher levels of pH2AX after oxaliplatin treatment in comparison to MSH3-proficient cells, suggesting that MSH3 plays an important role in repairing DNA double strand breaks (DSB). This role of MSH3 was further supported by our findings that MSH3-deficient cells were sensitive to olaparib, a Poly(ADP-ribose) polymerase inhibitor. Moreover, the combination of oxaliplatin and olaparib exhibited more than 10-fold increase in toxicity compared to either treatment individually. Conclusion:Collectively, our results provide novel evidence that MSH3 deficiency contributes to the cytotoxicity of platinum drugs through deficient DSB repair. These data lay foundation for the development of effective prediction and treatments for cancers with MSH3 deficiency. 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 4196. doi:10.1158/1538-7445.AM2011-4196

MSH3 Mediates Sensitization of Colorectal Cancer Cells to Cisplatin, Oxaliplatin, and a Poly(ADP-ribose) Polymerase Inhibitor

The MSH3 gene is one of the DNA mismatch repair (MMR) genes that has undergone somatic mutation frequently in MMR-deficient cancers. MSH3, together with MSH2, forms the MutSβ heteroduplex, which interacts with interstrand cross-links (ICLs) induced by drugs such as cisplatin and psoralen. However, the precise role of MSH3 in mediating the cytotoxic effects of ICL-inducing agents remains poorly understood. In this study, we first examined the effects of MSH3 deficiency on cytotoxicity caused by cisplatin and oxaliplatin, another ICL-inducing platinum drug. Using isogenic HCT116-derived clones in which MSH3 expression is controlled by shRNA expression in a Tet-off system, we discovered that MSH3 deficiency sensitized cells to both cisplatin and oxaliplatin at clinically relevant doses. Interestingly, siRNA-induced down-regulation of the MLH1 protein did not affect MSH3-dependent toxicity of these drugs, indicating that this process does not require participation of the canonical MMR pathway. Furthermore, MSH3-deficient cells maintained higher levels of phosphorylated histone H2AX and 53BP1 after oxaliplatin treatment in comparison with MSH3-proficient cells, suggesting that MSH3 plays an important role in repairing DNA double strand breaks (DSBs). This role of MSH3 was further supported by our findings that MSH3-deficient cells were sensitive to olaparib, a poly(ADP-ribose) polymerase inhibitor. Moreover, the combination of oxaliplatin and olaparib exhibited a synergistic effect compared with either treatment individually. Collectively, our results provide novel evidence that MSH3 deficiency contributes to the cytotoxicity of platinum drugs through deficient DSB repair. These data lay the foundation for the development of effective prediction and treatments for cancers with MSH3 deficiency.

Inhibition of the p38 MAPK pathway sensitises human colon cancer cells to 5-fluorouracil treatment

Colorectal cancer is the third most common cause of cancer-related deaths in the Western world. 5-Fluorouracil (5-FU) based chemotherapeutic regimes have been the mainstay of systemic treatment for disseminated colorectal cancer for many years. However, it only produces a 25% response rate due to the drug-resistance. The mitogen-activated protein kinase (MAPK) pathway is involved in the anti-apoptotic process; its activation provides cancer cells with a survival advantage to escape the apoptotic challenge. This study assessed whether the p38 MAPK pathway is involved in 5-FU resistance in colorectal cancer cells. 5-FU only or 5-FU combined with a p38 MAPK pathway inhibitor (SB203580) was used to treat 5-FU-resistant colorectal cancer cells. The effect of the treatment on cell viability, death and caspase activities was assessed. Western blotting was used to investigate the responses of apoptosis-related proteins following the treatment. Results showed that p38 MAPK inhibitor significantly increased colorectal cancer cell sensitivity to 5-FU. SB203580 in combination with 5-FU significantly reduced cell viability (P<0.01), and increased cell death and cellular caspase activity (P<0.01). Western blotting data revealed that SB203580 sensitises cancer cells to 5-FU due to an increase in Bax expression. These findings suggest that p38 MAPK is involved in cancer cell survival, and that the inhibition of p38 MAPK can enhance 5-FU to kill colorectal cancer cells.

Zerumbone, a Sesquiterpene from Southeast Asian Edible Ginger Sensitizes Colorectal Cancer Cells to Radiation Therapy

Zerumbone, a Sesquiterpene from Southeast Asian Edible Ginger Sensitizes Colorectal Cancer Cells to Radiation Therapy

The novel tumor suppressor HPP1 restores interferon sensitivity in colorectal cancer cells via activation of STAT 1 and 2 signaling pathways

The novel tumor suppressor HPP1 restores interferon sensitivity in colorectal cancer cells via activation of STAT 1 and 2 signaling pathways

Abstract A4: Identification of novel histone deacetylase inhibitor-regulated drug targets and a phase I/II trial of vorinostat in combination with 5-fluorouracil in metastatic colorectal cancer

Abstract Histone deacetylase inhibitors (HDACi) represent a novel class of agents that are undergoing clinical evaluation in colorectal cancer (CRC). Previously, we identified that HDACi including vorinostat (Vor) and LBH589 induced downregulation of the 5-FU target enzyme thymidylate synthase (TS) resulting in the sensitization of CRC cells to 5-FU. There were two goals in the present study: Firstly to identify additional genes in CRC cells that are differentially regulated by two clinically advanced HDACi, Vor and LBH589, furthering our understanding of their mechanism of action and to identify potential targets for novel HDACi-based drug combinations. Secondly, to extend our preclinical observations and evaluate the safety and feasibility of combining Vor with 5-FU in a phase I/II trial in patients with metastatic CRC (mCRC) and elevated intratumoral TS who had failed 5-FU-based therapy. HCT116 and HT29 CRC cells treated with Vor and LBH589 were analyzed using the Illumina Human-6 V2 BeadChip array. Differentially expressed genes (DEGs) were subject to hierarchical clustering, venn diagram analysis and interpreted using Ingenuity® Pathway Analysis. Results from microarray analysis were validated by quantitative PCR (qPCR) and analyzed in a panel of 8 CRC cell lines in vitro and in HCT116 and HT29 xenograft models. Bioinformatic analysis of the microarray results revealed significant similarity in DEGs following treatment with the two HDACi tested within each cell line. However, analysis of in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 DEGs modulated by both Vor and LBH589 were identified in both CRC cell lines analyzed and further validated in a panel of 8 CRC cell lines and in HCT116 and HT29 xenograft models treated with LBH589. This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel HDACi-based drug combinations in CRC. We concurrently conducted a Phase I/II clinical trial to determine the safety and feasibility of combining Vor with 5-FU in patients with mCRC and elevated intratumoral TS. Patients who had failed all standard therapeutic options were eligible. Intratumoral TS mRNA expression and peripheral blood mononuclear cell (PBMC) histone acetylation were measured before and after 6 consecutive days of Vor treatment at 400 mg PO daily. 5-FU/LV were given on days 6 and 7 and repeated every 2 wks, along with continuous daily Vor. Ten patients were enrolled and three dose levels were explored in the phase I portion of the study. Two dose-limiting toxicities (DLTs) were observed at the starting dose level, resulting in de-escalation to levels −1 and −2. Given the occurrence of two DLTs at each of the dose levels, we were unable to establish a maximum tolerated dose (MTD). Two patients achieved significant disease stabilization for 4 and 6 months. Grade 3 and 4 toxicities included fatigue, thrombocytopenia and mucositis. Intratumoral TS downregulation ≥50% was observed in one patient only. Acetylation of histone 3 was observed in PBMCs following Vor treatment. The study failed to establish a MTD and was terminated. The presence of PBMC histone acetylation indicates biological activity of Vor, however, consistent reductions in intratumoral TS mRNA were not observed. Alternate Vor dose-scheduling may alleviate the toxicity, achieve optimal TS downregulation and improve efficacy. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A4

Abstract C22: Identification of candidate microRNAs that correlate with sensitivity to the IGF-1R/IR inhibitor, OSI-906, in colorectal cancer (CRC) cell lines

Abstract Background: MicroRNAs (miRNAs) are small ∼20–22 nucleotide noncoding RNAs, thatwhich function as antisense regulators of gene expression by targeting specific mRNAs, and thereby modulate a variety of cellular signaling pathways. Deregulation of miRNA expression contributes to specific oncogenic events in human cancers and therefore may contribute to have a role in resistance to targeted cancer therapies. This study was based upon the hypothesis Here, we hypothesized that miRNA expression profiles wouldcan predict sensitivity to the novel IGF-1R tyrosine kinase inhibitor, OSI-906, in colorectal cancer (CRC) cell lines. Materials and Methods: To evaluate the sensitivity of CRC cells to OSI-906, a panel of 27 lines was exposed to increasing concentrations of the drug and assessed for proliferation using the sulforhodamine B assay. Six cell lines were sensitive (S, IC50 &amp;lt; 1.5 mol/L) and 21 were resistant (R, IC50 &amp;gt; 1.5 mol/L) to OSI-906. Baseline total RNA was isolated from 4 S and 4 R cell lines for miRNA and mRNA profiling on Dharmacon miRNA microarray and Affymetrix HG-U133 Plus 2.0 microarray platforms, respectively. For miRNA microarray data pre-processing, 342 miRNA probes with relative intensity of p ≤ 0.01 in at least 2 of the 8 experiments were kept for further data analysis. For mRNA microarray analysis, probe sets representing the same gene were collapsed based on maximum values and the expression levels were converted to a rank-based matrix. Anti-sense and mimic-miRNAs were used to study the effects of loss- and gain-of-function for the identified candidate miRNAs. Results: Significance Analysis of Microarray was used to determine the differentially expressed miRNAs in cell lines S or R to OSI-906 S vs. R cell lines profiles. Thirteen13 miRNAs were identified which showed a positive correlation with OSI-906 sensitivity. Three and 10 miRNAs were under- orand over-expressed in OSI-906 S cell lines, while respectively. gGene expression analysis revealed 78 and 42 genes under- orand over-expressed in OSI-906 S cell lines (p &amp;lt; 0.005), respectively. By comparing the predicted miRNA targets and differentially expressed genes, we identified two candidate miRNAs (hsa-miR-181a and hsa-miR-224) where the expression of their potential target genes, metallothioneins 1E and 2A (MT1E and MT2A) showed an inverse correlation with OSI-906 sensitivity. This observation was confirmed by qRT-PCR on both miRNAs and their target genes. To assess the loss- and gain-of-function effects of the candidate miRNAs, we delivered mimic- and anti-sense miRNAs to the OSI-906 R and S cell lines, respectively. Interestingly, over-expression of the miR-181a and miR-224 mimics in the OSI-906 R cell lines resulted in increased sensitivity to the drug, whereas. However, silencing of these miRNAs in the S cell lines did not demonstrateshow the reverse effect. Conclusion: These results suggest that loss of the candidate miRNA expression can confer OSI-906 resistance. Restoring the expression of these candidate miRNAs may induce sensitivity to OSI-906 in resistant CRC cell lines and may have clinical significant application in the targeted therapy of patients with advanced CRC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C22.

Abstract A39: Development and validation of an integrated genomic classifier to predict sensitivity to the IGF-1R/IR tyrosine kinase inhibitor, OSI-906, in colorectal cancer

Abstract Background: The role of the IGF-1 receptor (IGF-1R) pathway in mediating proliferative, invasive and pro-survival responses in cancer has led to a plethora of agents targeting this pathway, including antibodies and small molecule tyrosine kinase inhibitors. In order to maximize the clinical utility of such agents, it is imperative that patient selection strategies be applied. The goal of this study was to develop and characterize predictive biomarkers for the small molecule IGF-1R/IR tyrosine kinase inhibitor, OSI-906, in colorectal cancer (CRC). Methods and Results: Twenty-seven human CRC cell lines were exposed in vitro to OSI-906 and classified according to the IC50 value as sensitive (&amp;lt; 1.5 mol/L) or resistant (&amp;gt; 1.5 mol/L). Immunoblotting and immuno-histochemistry for effector proteins was performed on all CRC cell lines as well as fluorescent in-situ hybridization (FISH) to assess IGF-1R gene amplification and KRAS/BRAF/PI3K mutation sequencing. Baseline expression levels of effector proteins were not predictive of OSI-906 sensitivity. Although IGF-IR FISH did not reveal amplification, the OSI-906 sensitive CRC cell lines displayed an “unbalanced gain” of IGF-IR based upon ploidy. Likewise, there was a trend toward KRAS wildtype status and sensitivity to OSI-906. Next, the baseline gene expression of selected in vitro and in vivo sensitive or resistant cell lines was analyzed using Affymetrix U133 Plus 2.0 gene arrays. These transcriptional profiling data led to the development of a k-Top Scoring Pair (k-TSP) classifier which identified three gene pairs as the final classifier: (PROM1&amp;gt;MT1E), (LY75&amp;gt; OXCT1) and (HSD17B2&amp;gt;CALD1). Pathway analysis at baseline indicated IGF-IR signaling and insulin pathways were among the top 20 up regulated BioCarta pathways whereas in the resistant cells, the ErbB, MAPK, VEGF, and multiple cell adhesion/motility signaling pathways were prominently represented. From these data an integrated genomic classifier of FISH, KRAS status, and the k-TSP was developed and tested against 5 human CRC explants in vivo. The classifier was able to predict with 100% accuracy the responsiveness of these explants to OSI-906. Conclusions: These results indicate that an integrated approach to the development of individualized therapy is feasible and should be applied early in the development of this and other novel classes of anticancer agents. Additionally, such data may be used to develop rationale combination partners, such as VEGF or MEK inhibitors, to enhance responsiveness to OSI-906. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A39.

Abstract B169: Characterization of in vitro activity and predictive biomarkers of the novel p21-activated kinase 4 (PAK4) inhibitor, PF-3758309, in colorectal cancer cell lines

Abstract Background: The p21-activated kinase (PAK) family of serine/threonine kinases is overexpressed in multiple cancer types including breast, ovarian, colorectal, thyroid, and pancreatic. The two groups of PAK proteins, Group I (PAK 1–3) and Group II (PAK 4–6), are critical mediators of cell survival, motility, mitosis, transcription, and translation. The goal of this study was to assess the efficacy of a PAK4 inhibitor, PF-3758309, against a panel of colorectal cell lines and to identify potential predictive biomarkers of sensitivity or resistance that could be used to individualize therapy. Methods: A panel of 27 colorectal cancer (CRC) cell lines were exposed to increasing doses of PF-3758309 (0.015–1 µM) for 72 hours and analyzed for inhibition of proliferation using the sulforhodamine B (SRB) assay. Cell lines were designated sensitive (S), intermediate (I), or resistant (R) based on IC50's 0.125 and 1 µM, respectively. Assessment of induction of apoptosis was carried out using the caspase 3/7 assay at 24, 48, and 72 hours, whereas a soft agar colony assay was conducted on one sensitive and one resistant cell line to demonstrate anchorage- independent growth. To evaluate the effects on known target proteins of PAK-4, immunoblotting was performed for phosphorylated and total PAK-4, RhoA, GEF-H1, as well as proteins involved in apoptosis and the cell cycle. Lastly, to identify predictive genetic markers, five each of the most sensitive (IC50 1 mM) cell lines were subjected to gene array analysis using the Affymetrix U133 plus 2.0 gene chip. Results: Analysis of inhibition of proliferation in the 27 CRC cell lines demonstrated a greater than 10-fold (IC50 &amp;lt; 0.015 to &amp;gt; 1 mM) range of responsiveness to PF-3758309 that was accompanied by similarly diverse ranges of apoptosis induction. A sensitive cell line (HCT-116) demonstrated inhibition of anchorage-independent growth upon exposure to PF-3758309 that was not observed in the resistant (GEO) cell line. Overall, there did not appear to be an obvious relationship between effector protein expression and responsiveness to the PAK-4 inhibitor. Interestingly, gene array analysis indicated vimentin and ZEB1 expression to be increased, while E-cadherin was decreased, in the sensitive CRC cell lines. Other genes involved in cell cycle, MAPK pathway, and cytoskeleton dynamics were also differentially expressed. Conclusions: These data suggest that PAK4 inhibition by PF-3758309 results in efficacy against CRC cell lines as demonstrated by decreases in proliferation, anchorage-independent growth, and induction of apoptosis. The range of responsiveness to PF-3758309 and differential gene expression is being utilized to develop patient selective biomarkers for treatment with this novel compound. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B169.

Abstract A76: Characterization of CXC-chemokine expression in colorectal cancer tissue and its role in mediating chemoresistance of colorectal cancer cells to conventional and molecularly targeted therapies

Abstract Aim of study: The aims of this work were (i) to characterize CXC-chemokine and receptor expression in colorectal cancer (CRC) and (ii) study the role of CXC-chemokine signalling in modulating CRC cell sensitivity to Oxaliplatin and anti-EGFR therapy. Methods and Materials: CXC-chemokine and receptor expression was analysed in a retrospective study of colorectal cancer biopsy specimens in stage II and stage III CRC patients (n=254) who took part in a randomized phase III study comparing adjuvant 5FU chemotherapy with surgery alone between 1994 and 1997. For the in vitro work, studies were carried out in a panel of CRC cell lines comprising HCT116 clonal derivatives and LoVo cells. CXCL8 ligand and receptor expression was assessed by flow cytometry, qRT-PCR, western blotting and ELISA. Anti-apoptotic and EGFR signalling was also assessed using qRT-PCR and western blotting. NF-κB activity was assessed by electrophoretic mobility shift assay (EMSA) and Oxaliplatin response was assessed using cell counts. Results: Expression of CXCR1, CXCR2 and CXCL1 was elevated in tumor epithelium relative to the histologically normal adjacent colorectal cells (p&amp;lt;0.001, Wilcoxon signed ranks test). In addition, expression of CXCL1 correlated with the expression of its receptor CXCR2 in the tumor samples. In the CRC cell lines, constitutive expression of the ligands CXCL8 and CXCL1 was detected by immunoblotting and ELISA. Flow cytometry also confirmed the expression of the two receptors, CXCR1 and CXCR2 in these cell lines. The expression of the ligands and receptors was enhanced in response to Oxaliplatin treatment, demonstrated by qRT-PCR and immunoblotting. Furthermore, the administration of a CXCR2 antagonist AZ10397767 sensitized Oxaliplatin-promoted cytotoxicity and apoptosis in the PTEN-deficient HCT116 cells. Therefore, inhibition of CXC-chemokine signaling may modulate Oxaliplatin therapeutic response in PTEN-deficient CRC. Consistent with our published observations in prostate cancer cell lines, CXCL8 signalling increased NF-κB transcriptional activity and potentiated anti-apoptotic protein expression in CRC cells. Further studies demonstrated that CXCL8 signaling affects other survival pathways including activation of the Epidermal Growth Factor Receptor (EGFR). Immunoblotting confirmed that CXCL8 administration promotes post-translational regulation of EGFR through receptor phosphorylation in the CRC cells. Also, qRT-PCR experiments revealed that CXCL8 signaling potentiates the transcription of the EGFR ligands, Amphiregulin, and Transforming Growth factor-α (TGF-α) in CRC cell lines while ELISA detected an increased secretion of Amphiregulin following stimulation with CXCL8; this may play a role in the EGFR activation seen. Conclusions: Our studies suggest CRC malignant epithelium is subject to increased autocrine CXC-chemokine signaling compared to the adjacent normal colorectal tissue and this may reflect a role for the signaling in this disease. In-vitro work suggests that chemotherapy-induced CXC-chemokine signaling may underpin CRC chemoresistance through multiple pathways. These results support a role for CXC-chemokine signaling in the promotion of CRC and modulating sensitivity of this disease to current treatment Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A76.

Abstract B15: Targeted inhibition of mTOR signaling attenuates the growth and progression of colorectal cancer

Abstract Introduction: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the USA. The mammalian target of rapamycin (mTOR) acts downstream of PI3K to regulate cell growth, proliferation and survival. The mTOR kinase nucleates two distinct complexes, mTORC1 and mTORC2. The mTOR-RAPTOR complex (mTORC1) regulates translation initiation through the effectors S6K and 4E-BP1, while the mTOR-RICTOR-mSin1 complex (mTORC2) regulates the actin cytoskeleton and activates Akt, a key regulator of cell survival. Rapamycin inhibits the kinase activity of mTORC1; prolonged rapamycin treatment also inhibits mTORC2 assembly and Akt activation in certain cells. Furthermore, rapamycin mediated mTORC1 inhibition leads to loss of mTOR-S6K-IRS1 dependent feedback inhibition, thereby resulting in increased Akt activation. The purpose of the present study was to determine: (i) sensitivity of CRC cells to rapamycin treatment, and (ii) effect of targeting mTORC1 and mTORC2 upon CRC proliferation, apoptosis, cell cycle progression, migration, invasion and xenograft growth. Methods: HCT116, KM20, Caco-2 and SW480 human colon cancer cells were treated with rapamycin. Cells were also transfected with siRNA/shRNA directed against mTOR, RAPTOR or RICTOR. Effects on cell proliferation (measured by Coulter counter cell counts), apoptosis (measured by detecting the level of histone-associated DNA fragments), cell cycle progression (measured by fluorescence-activated cell sorting), migration (measured by wound healing assay), invasion (measured by transwell matrigel assay) and subcutaneous growth were analyzed. Results: (i) Immunohistochemical analysis showed that the mTORC1 and mTORC2 components, mTOR, RAPTOR and RICTOR are overexpressed in primary CRCs and matched liver metastases compared to normal colonic tissue. (ii) Treatment with rapamycin significantly decreased the proliferation of HCT116 and KM20 cells (rapamycin sensitive); however, rapamycin did not alter SW480 or Caco-2 proliferation (rapamycin resistant). (iii) Treatment with rapamycin significantly decreased the migration and invasion of HCT116 and SW480 cells. (iv) Transient siRNA-mediated knockdown of RAPTOR decreased proliferation of KM20 cells only, while knockdown of RICTOR decreased proliferation of all four cell lines. (v) Stable shRNA-mediated knockdown of mTORC1 and mTORC2 components leads to decreased proliferation, increased apoptosis and inhibition of G1-S phase cell cycle progression in HCT116 cells. (vi) Stable shRNA-mediated knockdown of mTORC1 and mTORC2 leads to decreased proliferation, but only mTORC2 knockdown leads to increased apoptosis in SW480 cells. (vii) Targeted inhibition of mTORC1 and mTORC2 attenuates growth of rapamycin-sensitive and — resistant CRCs in xenografted nude mice. Conclusions: The mTORC1 and mTORC2 components, mTOR, RAPTOR and RICTOR are overexpressed in CRCs. Transient inhibition of mTORC2, but not mTORC1, decreased the proliferation of rapamycin-sensitive and —resistant CRC cells. Stable inhibition of mTORC2 decreased proliferation and induced apoptosis in rapamycin-sensitive and —resistant CRC cells. Finally, targeted inhibition of both mTORC1 and mTORC2 attenuates subcutaneous growth of CRCs in vivo. Thus, inhibition of mTORC2 activity represents a novel therapeutic strategy for treatment of rapamycin-sensitive and, more importantly, rapamycin-resistant CRCs. Citation Information: Cancer Res 2009;69(23 Suppl):B15.

CIAP2 as a therapeutic target in colorectal cancer and other malignancies

Colorectal cancer is one of the most common malignancies worldwide and 70% of tumors are resectable, but patients with metastatic diseases cannot be cured with current treatment modalities. Inhibition of the apoptotic pathway is one of the factors that may be responsible for carcinogenesis and drug resistance, and the inhibitor of apoptosis protein (IAP) family is thought to prevent apoptosis through inhibition of direct caspases and pro-caspases. Recently an increasing amount of evidence has been accumulated regarding cIAP2 and other IAP proteins of the antiapoptotic pathway and NF-κB signal transduction. IAPs are abnormally regulated and expressed in the majority of human malignancies at elevated levels. As a result, they have recently been reported to be therapeutic targets. The downregulation of cIAP2 efficiently enhances apoptosis through the activation of caspase 3/7 and 5-fluorouracil (5-FU) sensitivity in colorectal cancer cells exposed to 5-FU. This report reviews the evidence for cIAP2 and other IAP molecules as a therapeutic target for malignancies including colorectal cancer. So far, the information on colorectal cancer is limited; so this study includes other malignancies as well, in order to summarize the current knowledge of drug development targeting IAP molecules and provide an overview of the future course.

W1926 Collagen IV-β1 Integrin Interaction Modulates PI3K/AKT and Influences Chemotherapy Sensitivity in Colorectal Cancer Cells in the Presence of SPARC

W1926 Collagen IV-β1 Integrin Interaction Modulates PI3K/AKT and Influences Chemotherapy Sensitivity in Colorectal Cancer Cells in the Presence of SPARC