Therapeutic Approach For Colorectal Cancer Research Articles

Suppression of Nanog inhibited cell migration and increased the sensitivity of colorectal cancer cells to 5-fluorouracil

Nanog is a major transcription factor related to cellular multipotency that plays important roles in the development of tumor cells, drug resistance, migration, and stemness; indicating its great potential as a therapeutic target for various malignancies including colorectal cancer (CRC). Therefore, this study was aimed to evaluate the Nanog suppression effect using small interference RNA (siRNA) combined with 5-fluorouracil (5-FU) on CRC cells. Nanog-overexpressing SW-480 cells were transfected with Nanog si-RNA and treated with 5-FU, in combination or separately. Subsequently, it was observed that Nanog expression was significantly reduced after transfection of SW-480 cells using Nanog siRNA in mRNA and protein levels. Furthermore, Nanog knockdown significantly increased CRC cell sensitivity to 5-FU drug via modulating Bax and Bcl-2 mRNA expression. Also, Nanog knockdown and 5-FU treatment cooperatively decreased the migration and self-renewal ability of SW-480 cells by regulating the expression of relevant genes. Moreover, combination therapy led to cell cycle arrest at the sub-G1 phase in CRC cells. In conclusion, our results indicated that Nanog may play an important role in the drug sensitivity, migration, and self-renewal of CRC cells; suggesting Nanog as a promising target in combination with 5-FU for the development of new therapeutic approaches for CRC.

MiR-27a-3p regulates proliferation and apoptosis of colon cancer cells by potentially targeting BTG1.

microRNA (miR/miRNA)-27a-3p has been reported to be abnormally expressed in various types of cancer, including colorectal cancer (CRC). B-cell translocation gene 1 (BTG1) has also been implicated with CRC. However, the association between miR-27a-3p and BTG1 in CRC, to the best of our knowledge, has not been investigated. In order to assess whether miR-27a-3p is associated with CRC, reverse transcription-quantitative PCR was performed on 20 paired CRC and paracancerous tissues for miRNA analysis. For the screening and validation of miR-27a-3p expression in colon cancer, several colon cancer cell lines (HCT-116, HCT8, SW480, HT29, LOVO and Caco2) and the normal colorectal epithelial cell line NCM460 were examined. The highest expression levels of miR-27a-3p were detected in the HCT-116, which was selected for further experimentation. The HCT-116 cells were divided into control, miR-27a-3p mimic and inhibitor groups, and cell proliferation was tested using an MTT assay. Additionally, miR-27a-3p inhibitor/mimic or BTG1 plasmid were transfected into the HCT-116 cells, and flow cytometry was performed to analyze cell cycle distributions. TUNEL analysis was performed to detect apoptosis. Protein levels of factors in the downstream signaling pathway mediated by miR-27a-3p [ERK/mitogen-activated extracellular signal-regulated kinase (MEK)] were detected. miR-27a-3p was revealed to be overexpressed in human CRC tissues and colon cancer cell lines. Knockdown of miR-27a-3p suppressed proliferation of HCT-116 cells and apoptosis was increased. It further markedly upregulated expression levels of BTG1 and inhibited activation of proteins of the ERK/MEK signaling pathway. In addition, overexpression of BTG1 in HCT-116 cells triggered G1/S phase cell cycle arrest and increased apoptosis via the ERK/MEK signaling pathway. In conclusion, the present study demonstrated that the effects of miR-27a-3p on colon cancer cell proliferation and apoptosis were similar to those of the tumor suppressor gene BTG1. The miR-27a-3p/BTG1 axis may have potential implications for diagnostic and therapeutic approaches in CRC.

Revisiting the Role of Exosomes in Colorectal Cancer: Where Are We Now?

Exosomes (Exos) are nano-sized extracellular vesicles constitutively released by both prokaryotic and eukaryotic cells. Their role as inter-cellular messengers involved in both physiological and pathological processes has overwhelmingly come to light in the last decade, and their contribution to cancerogenesis and tumor metastasis is under intensive investigation. Here we review the most recent information concerning Exos in colorectal cancer (CRC) and focus on their effects on tumor microenvironment and the immune system, as well as unravel their role in the formation of the pre-metastatic niche and in drug resistance. Such a recent knowledge on Exos depicts their potential translations into the clinical arena, either as an alternative tool of “liquid biopsy” or novel therapeutic approaches for CRC. However, due to the limited data available from clinical trials, they need further validations before addressing their putative application in oncology.

PO-203 A novel member in the β-catenin destruction complex: may MAPK14/P38α foster new therapeutic approaches in colorectal cancer?

IntroductionOne of the most commonly deregulated signalling pathways in colorectal cancer (CRC) is the Wnt cascade, which is controlled by APC. APC regulates β-catenin levels, thereby modulating the transcriptional activity of the TCF/LEF transcription factors. High levels of nuclear β-catenin lead to constitutive activation of the Wnt pathway, loss of normal cellular architecture and neoplastic transformation. Previous reports indicate that Wnts are capable of activating p38 MAPKs. A few older studies carried out in other tissues provided evidence that Wnt3a could activate p38 suggesting that the p38 pathway may feed into the canonical Wnt/β-catenin pathway at least at the level of GSK3β. We recently showed that p38α is required to maintain CRC metabolism and survival, as its inhibition leads to activation of FoxO3A, autophagy, cell death and tumour growth reduction both in vitro and in vivo.Material and methodsWe performed extensive characterisation of the functional interaction between p38 and the APC/β-catenin/GSK3β complex (co-localization analysis by confocal microscopy and co-immunoprecipitation studies) in several cell lines in vitro and in the APCMin/+ mouse preclinical model in vivo.Results and discussionsOur data showed that CRC cells have higher levels of activated p38 than their normal counterparts, and experiments using kinase-specific inhibitors revealed that these cells are ‘addicted’ to p38 activity. Interestingly, p38α blockade reduced the size and number of adenomas in the small bowel of APCMin/+ mice. Significant results were obtained in vivo by co-immunoprecipitation analysis of tissues from normal mice and APCMin/+ mice treated or not with AOM. Our findings confirmed the presence of p38α in APC/β-catenin/GSK3β complexes in CRC cells. Importantly, p38α co-localised with β-catenin in both normal and cancer cells; however, these proteins were confined to the cytoplasm in colonocytes, while they occupied discrete nuclear regions in CRC cells. These data were further corroborated by the inhibitory effect of p38α blockade on β-catenin-responsive genes (i.e. c-Myc, cyclin D1/2). Characterisation of this novel functional interaction was also extended with chromatin immunoprecipitation experiments.ConclusionIdentification of p38α as a novel member of the APC/β-catenin/GSK3β complex could help elucidate mechanisms contributing to human colon tumour pathogenesis and allow for the development of new strategies for CRC treatment.

Revisiting colorectal cancer animal model – An improved metastatic model for distal rectosigmoid colon carcinoma

Colorectal cancer (CRC) is the second most frequent and fatal cancer in Western countries. Understanding its biology with different incidence along the colon and rectum, genetic profile and how these factors contribute to local/distant progression, has been hampered by the lack of a suitable CRC model.We report a reproducible model, using human CRC cell lines (CL) (WiDr, LS1034, C2BBe1) injected (1 × 107 cells/animal) in RNU rats (n = 55) which underwent cecostomy and descending colostomy with mucosal-cutaneous fistula of the sigmoid colon. CL were characterized by immunohistochemistry: CK20, CDX2, P53, vimentin, Ki67, CD44, CD133, E-cadherin, β-catenin and CEA; cancer stem cells-immune system interaction was studied and tumor progression was assessed with nuclear medicine imaging (99mTc-MIBI).Animals developed locally invasive tumors and with WiDr neural invasion was registered. Cancer stem cells were detected in WiDr (CD44 positive). All the cell lines stimulated the immune system, being WiDr the most aggressive. Imaging studies demonstrated tumor uptake.With this CRC model we can study the microenvironment role and tumor-stroma interactions. All CL developed primary disease, but only the WiDR established neural invasion which may represent a metastatic pathway. This model can help unveiling the underlying metastatic mechanisms, and ultimately test better therapeutic approaches for CRC.

81P - Simultaneous use of erythropoietin and LFM-A13 as a new therapeutic approach for colorectal cancer

81P - Simultaneous use of erythropoietin and LFM-A13 as a new therapeutic approach for colorectal cancer

Simultaneous use of erythropoietin and LFM-A13 as a new therapeutic approach for colorectal cancer.

Background and PurposeBruton's tyrosine kinase (Btk) is a non‐receptor tyrosine kinase involved in the activation of signalling pathways responsible for cell maturation and viability. Btk has previously been reported to be overexpressed in colon cancers. This kind of cancer is often accompanied by anaemia, which is treated with an erythropoietin supplement. The goal of the present study was to assess the effects of combination therapy with erythropoietin β (Epo) and LFM‐A13 (Btk inhibitor) on colon cancer in in vitro and in vivo models.Experimental ApproachDLD‐1 and HT‐29 human colon adenocarcinoma cells were cultured with Epo and LFM‐A13. Cell number and viability, and mRNA and protein levels of Epo receptors, Btk and Akt were assessed. Nude mice were inoculated with adenocarcinoma cells and treated with Epo and LFM‐A13.Key ResultsThe combination of Epo and LFM‐A13 mostly exerted a synergistic inhibitory effect on colon cancer cell growth. The therapeutic scheme used effectively killed the cancer cells and attenuated the Btk signalling pathways. Epo + LFM‐A13 also prevented the normal process of microtubule assembly during mitosis by down‐regulating the expression of Polo‐like kinase 1. The combination of Epo and LFM‐A13 significantly reduced the growth rate of tumour cells, while it showed high safety profile, inducing no nephrotoxicity, hepatotoxicity or changes in the haematological parameters.Conclusion and ImplicationsEpo significantly enhances the antitumour activity of LFM‐A13, indicating that a combination of Epo and LFM‐A13 has potential as an effective therapeutic approach for patients with colorectal cancer.

Targeting the PI3K Signalling as a Therapeutic Strategy in Colorectal Cancer.

Colorectal cancer (CRC) remains one of the leading causes of cancer mortality worldwide. Regarded as a heterogeneous disease, a number of biomarkers have been proposed to help in the stratification of CRC patients and to enable the selection of the best therapy for each patient towards personalized therapy. However, although the molecular mechanisms underlying the development of CRC have been elucidated, the therapeutic strategies available for these patients are still quite limited. Thus, over the last few years, a multitude of novel targets and therapeutic strategies have emerged focusing on deregulated molecules and pathways that are implicated in cell growth and survival. Particularly relevant in CRC are the activating mutations in the oncogene PIK3CA that frequently occur in concomitancy with KRAS and BRAF mutations and that lead to deregulation of the major signalling pathways PI3K and MAPK, downstream of EGFR. This review focus on the importance of the PI3K signalling in CRC development, on the current knowledge of PI3K inhibition as a therapeutic approach in CRC and on the implications PI3K signalling molecules may have as potential biomarkers and as new targets for directed therapies in CRC patients.

Abstract 452: Activation of Akt pathway and autophagy promotes resistance to FASN inhibition in colorectal cancer patient-derived xenograft models

Abstract Fatty Acid Synthase (FASN), a key enzyme of de novo lipogenesis, is upregulated in many cancers including colorectal cancer (CRC); increased FASN expression is associated with poor prognosis. Potent FASN inhibitors developed by 3-V Biosciences demonstrate anti-tumor activity in vitro and in vivo and a favorable tolerability profile in a Phase I clinical trial in solid tumor patients. However, CRC characteristics associated with responsiveness to FASN inhibition are not fully understood. The purpose of our study was: (i) to determine the effect of FASN inhibition on tumor growth in CRC patient-derived xenografts (PDXs); (ii) to identify potential biomarkers associated with CRC responsiveness to FASN inhibition; and (iii) to explore new combination strategies with FASN inhibitors. METHODS. Tumor growth was assessed in 9 PDXs established in NSG mice using freshly resected specimens. Once the xenografts grew to ~100 mm3, mice were randomized into two groups (n=5) to receive either vehicle or TVB-3664 or four groups (n=10) for TVB-3664 treatment in combination with either MK2206 or Chloroquine (CQ). Tumor volume and animal weights were measured weekly. Western blot analysis and immunohistochemistry staining were used to identify FASN-mediated changes in signaling pathways. Changes in metabolites and lipids were analyzed by nuclear magnetic resonance and mass spectrometry in plasma and tumor tissues. Next Generation Sequencing was used to assess the mutation profile of 198 oncogenes in patient tumors and PDXs. RESULTS. PDXs showed a wide range of sensitivity to FASN inhibition: TVB-3664 treatment attained significant response (reduced tumor volume) in 3 PDXs, significant response followed by developed resistance in one PDX, and no response in 5 PDXs. Activation of Akt and AMPK pathways was associated with resistance to FASN inhibition and combination of TVB-3664 with either MK2206 or CQ led to a significant reduction in tumor volume as compared to either drug alone. Moreover, TVB-3664 treatment significantly decreased the total palmitate level in plasma and the levels of triglycerides, diglycerides, phosphatidylserines, phosphatidylethanolamines, and phosphatidylcholines in tumor tissues. Furthermore, a significant decrease in the levels of AXP-1, AXP-2 and myo-Inositol-2 was observed in tumors responsive to FASN inhibition. CONCLUSIONS. Our studies demonstrate that TVB-3664 shows anti-tumor activity in CRC. Importantly, our results suggest that activation of Akt and autophagy are major mechanisms of resistance to FASN inhibition and demonstrate that combine inhibition of these pathways and FASN may be a new therapeutic approach in CRC. Ongoing studies of correlation between mutation and metabolic profiles of tumors and tumor response to FASN inhibition aim to identify a subset of CRC patients that are likely to respond to FASN-targeted therapy. Citation Format: Yekaterina Y. Zaytseva, Piotr G. Rychahou, Anh-Thu Le, Robert M. Flight, Timothy L. Scott, Jennifer W. Harris, Sally Hodges, Brent J. Hallahan, Dana L. Napier, Jinpeng Liu, Chi Wang, Manjula Sunkara, Andrew Morris, Ji Tae Kim, Sivakumaran Theru Arumugam, Andrew Lane, Teresa W. Fan, Hunter Moseley, Tianyan Gao, Eun Y. Lee, Heidi L. Weiss, Timothy S. Heuer, George Kemble, B Mark Evers. Activation of Akt pathway and autophagy promotes resistance to FASN inhibition in colorectal cancer patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 452. doi:10.1158/1538-7445.AM2017-452

Targeting the tumor microenvironment as a potential therapeutic approach in colorectal cancer: Rational and progress

Colorectal cancer (CC) is often diagnosed at a late stage when tumor metastasis may have already occurred. Current treatments are often ineffective in metastatic disease, and consequently late diagnosis is often associated with poor outcomes in CC. Alternative strategies are therefore urgently required. An interaction between epithelial cancer cells and their tissue microenvironment is a contributor to metastasis, and therefore recent studies are beginning to focus on the properties of the tumor microenvironment and the mechanism by which the metastatic cells exploit the tumor microenvironment for survival, immune evasion, and growth. We have reviewed the development of the combined therapeutic approaches that have focused on targeting the microenvironment of CC.

Dual inhibition of BDNF/TrkB and autophagy: a promising therapeutic approach for colorectal cancer.

Colorectal cancer (CRC) is the most common digestive cancer in the Western world. Despite effective therapies, resistance and/or recurrence frequently occur. The present study investigated the impact of two survival pathways—neurotrophic factors (TrkB/BDNF) and autophagy—on cell fate and tumour evolution. In vitro studies were performed on two CRC cell lines, SW480 (primary tumour) and SW620 (lymph node invasion), which were also used for subcutaneous xenografts on a nude mouse model. In addition, the presence of neurotrophic factors (NTs) and autophagy markers were assessed in tissue samples representative of different stages. On the basis of our previous study (which demonstrated that TrkB overexpression is associated with prosurvival signaling in CRC cells), we pharmacologically inhibited NTs pathways with K252a. As expected, an inactivation of the PI3K/AKT pathway was observed and CRC cells initiated autophagy. Conversely, blocking the autophagic flux with chloroquine or with ATG5‐siRNA overactivated TrkB/BDNF signaling. In vitro, dual inhibition improved the effectiveness of single treatment by significantly reducing metabolic activity and enhancing apoptotic cell death. These findings were accentuated in vivo, in which dual inhibition induced a spectacular reduction in tumour volume following long‐term treatment (21 days for K252a and 12 days for CQ). Finally, significant amounts of phospho‐TrkB and LC3II were found in the patients’ tissues, highlighting their relevance in CRC tumour biology. Taken together, our results show that targeting NTs and autophagy pathways potentially constitutes a new therapeutic approach for CRC.

Chemotherapy-induced nausea and vomiting (CINV) in 190 colorectal cancer patients: a prospective registration study by the CINV study group of Japan

ABSTRACTObjectives: Chemotherapy is an indispensable therapeutic approach for colorectal cancer both in the adjuvant and metastatic setting. Although chemotherapy-induced nausea and vomiting (CINV) is one of the most crucial adverse events, many aspects of CINV in patients with colorectal cancer remain unclear.Methods: This multicenter, prospective, observational study analyzed the data of 190 colorectal cancer patients scheduled for moderately emetogenic chemotherapy (MEC). The patients recorded the incidence of CINV and severity of nausea by visual analogue scales daily for 7 days after receiving chemotherapy.Results: All 190 patients received MEC and 99% of patients received antiemetic therapy in compliance with guidelines. Acute CINV was well controlled. 13 (6.8%) patients suffered from acute nausea and 4 (2.1%) experienced acute vomiting, whereas the prevalence of delayed CINV was relatively high. Delayed nausea occurred in 71 (37.4%) patients and delayed vomiting in 24 (12.6%). History of motion sickness was a significant independent risk factor for delayed nausea (Odd ratio 3.89, 95% confidence interval 1.49–10.19, p = 0.0056).Conclusions: The compliance with CINV guidelines in colorectal cancer chemotherapy was quite high and led to good control of chemotherapy-induced vomiting in Japan. However, the incidence of delayed nausea remained high in patients receiving MEC.

Novel biotechnology approaches in colorectal cancer diagnosis and therapy.

With ever-increasing molecular information about colorectal cancer (CRC), there is an expectation to detect more sensitive and specific molecular markers for new advanced diagnostic methods that can surpass the limitations of current screening tests. Moreover, enhanced molecular pathology knowledge about cancer has led to the development of targeted therapies, designed to interfere with specific aberrant biological pathways in cancer. Furthermore, biotechnology has opened a new window in CRC diagnosis and treatment by introducing different application of antibodies, antibody fragments, non-Ig scaffold proteins, and aptamers in targeted therapy and drug delivery. This review summarizes the molecular diagnostic and therapeutic approaches in CRC with a focus on genetic and epigenetic alterations, protein and metabolite markers as well as targeted therapy and drug delivery by Ig-scaffold proteins, non-Ig scaffold proteins, nanobodies, and aptamers.

Abstract B54: The role of fatty acid synthase in regulation of autophagy in colorectal cancer

Abstract Fatty Acid Synthase (FASN), a key enzyme of de novo lipid synthesis, is significantly up regulated in colorectal cancer (CRC) and its activity is associated with worse prognosis. Recent studies suggest that up regulation of FASN provides a survival advantage to cancer cells by reprogramming metabolic pathways and maintaining energy homeostasis. Autophagy is an adaptive survival mechanism induced under conditions of stress. However, the interconnection between de novo lipogenesis and autophagy has not been studied. Therefore, the purpose of our study was to determine the role of FASN in regulation of autophagy in vitro and in human CRC. Methods: Expression of p62, LC3, and Beclin1 (autophagy markers) and pAMPK was assessed in CRC cell lines with altered expression of FASN cultured in normal, serum free medium and/or detached conditions by western blot and confocal microscopy. Human specimens, including matching cancer, normal colon and liver metastasis tissues from 19 patients were analyzed for expression of FASN, p62, and pAMPK by immunohistochemistry (IHC). The effect of stable knockdown of p62 on proliferation and apoptosis was analyzed in CRC cell lines by cell counting, cell death ELISA and western blot analysis. Results: We demonstrated that under metabolic stress conditions overexpression of FASN is associated with a decrease in autophagy as shown by accumulation of p62, a marker of inhibition of autophagy, and a decrease in LC3, an autophagosome marker, in CRC cell lines. Under energy stress conditions, overexpression of FASN is also associated with a decrease in activation of pAMPK. Statistical analysis of IHC staining showed that expression of FASN and p62 are significantly higher in cancer and metastatic tissues as compared to normal colon tissues. A significant moderate correlation between expression of FASN and p62 was determined in human primary CRCs. Stable knockdown of p62 in CRC cell lines demonstrated that an increased level of p62 is associated with increased cellular proliferation and decreased cell death. Conclusions: We have identified a novel link between FASN and autophagy, and showed that, under metabolic stress conditions, overexpression of FASN is associated with inhibition of autophagy. Furthermore, for the first time, we showed the significant correlation between expression of FASN and p62 in human CRC. The effects of p62 on cellular proliferation and apoptosis suggest that p62 may also play a role independent of autophagy; therefore, further studies are require to understand how FASN-mediated up regulation of p62 contributes to tumorigenesis. The differential expression of FASN in normal versus cancer cells makes de novo lipogenesis a desirable target for therapeutic intervention. Understanding the role of FASN in regulation of survival mechanisms of cancer cells including autophagy will reveal new therapeutic approaches in CRC. Citation Format: Yekaterina Zaytseva, Jennifer Harris, Ji Tae Kim, Eun Lee, Heidi Weiss, Tianyan Gao, Mark Evers. The role of fatty acid synthase in regulation of autophagy in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B54.

Therapeutic targets in the Wnt signaling pathway: Feasibility of targeting TNIK in colorectal cancer

The genetic and epigenetic alterations occurring during the course of multistage colorectal carcinogenesis have been extensively studied in the last few decades. One of the most notable findings is that the great majority of colorectal cancers (>80%) have mutations in the adenomatous polyposis coli (APC) tumor suppressor gene. Loss of functional APC protein results in activation of canonical Wnt/β-catanin signaling and initiates intestinal carcinogenesis. Mutational inactivation of APC is the first genetic event, but colorectal cancer cells retain their dependency on constitutive Wnt signal activation even after accumulation of other genetic events. Accordingly, pharmacological blocking of Wnt signaling has been considered an attractive therapeutic approach for colorectal cancer. Several therapeutics targeting various molecular components of the Wnt signaling pathway, including porcupine, frizzled receptors and co-receptor, tankyrases, and cAMP response element binding protein (CREB)-binding protein (CBP), have been developed, and some of those are currently being evaluated in early-phase clinical trials. Traf2- and Nck-interacting protein kinase (TNIK) has been identified as a regulatory component of the T-cell factor-4 and β-catenin transcriptional complex independently by two research groups. TNIK regulates Wnt signaling in the most downstream part of the pathway, and its inhibition is expected to block the signal even in colorectal cancer cells with APC gene mutation. Here we discuss some of the TNIK inhibitors under preclinical development.

Frameshift mutation of UVRAG: Switching a tumor suppressor to an oncogene in colorectal cancer

Colorectal cancer (CRC) ranks as the second leading cause of cancer-related deaths in the Western world. It has a nearly 50% metastasis rate and only a subset of patients respond to current treatment strategy. UVRAG, a key autophagy effector and a guardian of chromosomal stability, is truncated by a frameshift (FS) mutation in CRC with microsatellite instability (MSI). However, the pathological and clinical significance of this UVRAG truncation remains less understood. Our recent study discovered that this FS mutation yields a much shortened form of the UVRAG protein, which counteracts most of the tumor-suppressor functions of wild-type (WT) UVRAG in autophagy, centrosome stability, and DNA repair in a dominant-negative fashion. Whereas this truncated mutation of UVRAG promotes tumorigenesis, epithelial-to-mesenchymal transition, and metastasis, it appears to sensitize CRC tumors to adjuvant chemotherapy, making it a potential molecular marker to individualize therapeutic approach in CRC.

Colorectal cancer-related mutant KRAS alleles function as positive regulators of autophagy.

The recent interest to modulate autophagy in cancer therapy has been hampered by the dual roles of this conserved catabolic process in cancer, highlighting the need for tailored approaches. Since RAS isoforms have been implicated in autophagy regulation and mutation of the KRAS oncogene is highly frequent in colorectal cancer (CRC), we questioned whether/how mutant KRAS alleles regulate autophagy in CRC and its implications. We established two original models, KRAS-humanized yeast and KRAS-non-cancer colon cells and showed that expression of mutated KRAS up-regulates starvation-induced autophagy in both. Accordingly, KRAS down-regulation inhibited autophagy in CRC-derived cells harboring KRAS mutations. We further show that KRAS-induced autophagy proceeds via up-regulation of the MEK/ERK pathway in both colon models and that KRAS and autophagy contribute to CRC cell survival during starvation. Since KRAS inhibitors have proven difficult to develop, our results suggest using autophagy inhibitors as a combined/alternative therapeutic approach in CRCs with mutant KRAS.

Abstract 1151: Overexpression of Fatty Acid Synthase is associated with inhibition of autophagy under conditions of metabolic stress in colorectal cancer cells

Abstract Fatty Acid Synthase (FASN), a key enzyme of de novo lipid synthesis, is significantly up regulated in colorectal cancer (CRC) and its activity is associated with poor prognosis. Recent studies suggest that FASN plays a crucial role in the survival of cancer cells by reprogramming metabolic pathways to maintain energy homeostasis under conditions of metabolic stress. However, the mechanisms of metabolic adaptation of cancer cells regulated by FASN to promote their survival are not yet fully understood. Cancer cells use autophagy as one of the survival strategies under metabolic stress. The purpose of our study was to determine the role of aberrant activation of FASN in regulation of autophagy in vitro and in human CRC. METHODS. Expression of p62 and LC3 (autophagy markers), pAMPK, and Cyclin D was assessed in CRC cell lines with altered expression of FASN cultured in normal, serum free medium and/or detached conditions by Western blot and confocal microscopy. Human specimens, including matching cancer, normal colon and liver metastasis tissues from 19 patients were analyzed for expression of FASN, p62, and pAMPK by immunohistochemistry (IHC). RESULTS. We demonstrated that under metabolic stress conditions overexpression of FASN is associated with a decrease in autophagy as shown by accumulation of p62, a marker of inhibition of autophagy, and a decrease in LC3A, an autophagosome marker, in CRC cell lines. Under energy stress conditions, overexpression of FASN is also associated with an increase in Cyclin D and a decrease in activation of pAMPK. Statistical analysis of IHC staining showed that expression of FASN, p62, and pAMPK is significantly higher in cancer and metastatic tissues as compared to normal colon tissues. Using Spearman's rank correlation, we determined moderate correlation between expression of FASN and p62 in human primary CRC and liver metastasis. Similar results were obtained using Western blot analysis on human normal colon and matching cancer tissues. CONCLUSIONS. Our data suggest that overexpression of FASN is protective during metabolic stress conditions by shifting metabolism towards higher energy production as indicated by activity of pAMPK. Furthermore, we have identified a novel link between FASN and p62, and showed that, under metabolic stress conditions, overexpression of FASN is associated with inhibition of autophagy. However, further studies are require to understand how FASN-mediated up regulation of p62 contributes to tumorigenesis. The differential expression of FASN in normal versus cancer cells makes de novo lipogenesis a desirable target for therapeutic intervention. Understanding the role of FASN in regulation of survival and aggressive behavior of cancer cells will uncover new therapeutic approaches in CRC. Citation Format: Yekaterina Y. Zaytseva, Jennifer W. Harris, Ji Tae Kim, Tianyan Gao, Eun Y. Lee, Heidi L. Weiss, B Mark Evers. Overexpression of Fatty Acid Synthase is associated with inhibition of autophagy under conditions of metabolic stress in colorectal cancer cells. [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 1151. doi:10.1158/1538-7445.AM2015-1151

Ectopic expression of p33ING1b suppresses proliferation and induces apoptosis in colonic adenocarcinoma cells.

Inhibitor of growth 1b (ING1b) is considered to be a class II tumor suppressor gene. Although decreased expression of p33ING1b has previously been reported in colorectal cancer (CRC), its role in CRC has remained to be elucidated. The present study was designed to assess the function of p33ING1b in CRC and to further evaluate its underlying mechanisms of action. Western blot analysis confirmed that ING1b gene expression was significantly decreased in CRC tissues compared with that of adjacent non-tumorous colorectal tissues. Furthermore, recombinant adenovirus-mediated ectopic expression of p33ING1b resulted in growth inhibition, G1-phase cell cycle arrest and apoptosis in the SW480, HT29 and LoVo colorectal adenocarcinoma cell lines. The results suggested that the downregulation of ING1b contributes to colorectal carcinogenesis and that ectopic expression of ING1b may be a potentially useful therapeutic approach for CRC.

AXL is an oncotarget in human colorectal cancer.

AXL is a tyrosine kinase receptor activated by GAS6 and regulates cancer cell proliferation migration and angiogenesis. We studied AXL as new therapeutic target in colorectal cancer (CRC). Expression and activation of AXL and GAS6 were evaluated in a panel of human CRC cell lines. AXL gene silencing or pharmacologic inhibition with foretinib suppressed proliferation, migration and survival in CRC cells. In an orthotopic colon model of human HCT116 CRC cells overexpressing AXL, foretinib treatment caused significant inhibition of tumour growth and peritoneal metastatic spreading. AXL and GAS6 overexpression by immunohistochemistry (IHC) were found in 76,7% and 73.5%, respectively, of 223 human CRC specimens, correlating with less differentiated histological grading. GAS6 overexpression was associated with nodes involvement and tumour stage. AXL gene was found amplified by Fluorescence in situ hybridization (FISH) in 8/146 cases (5,4%) of CRC samples. Taken together, AXL inhibition could represent a novel therapeutic approach in CRC.