CRC Therapy Research Articles

CCNA2 acts as a novel biomarker in regulating the growth and apoptosis of colorectal cancer.

ObjectiveColorectal cancer (CRC) is considered to be the most prevalent malignant tumors that contribute to high cancer-related mortality. However, the signaling pathways involved in CRC and CRC-driven genes are largely unknown. We seek to discover a novel biomarker in CRC.Materials and methodsAll clinical CRC samples (n=33) were from Xiangya Hospital. We first selected CCNA2 by integrated bioinformatics analysis of four GSE databases. Next, the expression of CCNA2 in tissues and cell lines was verified by quantitative real-time PCR. The effects of CCNA2 on cell growth, proliferation, cell cycle, and apoptosis were examined by in vitro assays.ResultsWe identified 498 shared DEGs (294 upregulated and 204 downregulated), and the top ten hub genes were selected by integrated analysis. These hub genes were significantly overexpressed in CRC samples and were positively correlated. Our data revealed that the expression of CCNA2 in CRC tissues is higher than that in normal tissues. The CCNA2 knockdown could significantly suppress CRC cell growth by impairing cell cycle progression and inducing cell apoptosis.ConclusionCCNA2, as a novel oncogenic gene, plays a role in regulating cancer cell growth and apoptosis. It could be used as a new biomarker for diagnosis and therapy in CRC.

FOXD4 induces tumor progression in colorectal cancer by regulation of the SNAI3/CDH1 axis

ABSTRACTColorectal cancer (CRC) is ranked third as the most common malignancy, and it develops into metastasis at a high rate. Importantly, distant metastasis is considered to be a key factor for colorectal therapy. In the present study, we identified FOXD4, a transcription factor belonging to the forkhead/winged helix-box (FOX) family, as a novel biomarker for diagnosis and treatment of patients with CRC. We revealed that FOXD4 was up-regulated in CRC tissues and increased the metastatic ability of CRC cells. Additionally, FOXD4 affected the metastasis of CRC by inducing the epithelial-mesenchymal transition (EMT) process. Furthermore, FOXD4 could directly bind the SNAI3 promoter during EMT in CRC and then facilitate CRC metastasis. In summary, the present research strongly suggests that FOXD4 is a valuable marker for CRC, and that targeting FOXD4 may be a novel strategy for enhancing the treatment outcomes of CRC therapy

Abstract 5480: Colorectal cancer cell lines harboring mutation in Wnt/β-catenin pathway shows enhanced sensitivity to the stearoyl-CoA desaturase (SCD) inhibitor, DSP-0692

Abstract Colorectal cancer (CRC) is one of the most frequent malignant diseases worldwide. Recent studies suggest that a small subpopulation of cells known as cancer stem cells (CSCs) may promote the high metastasis and relapse associated with CRC. It has been reported that Wnt/β-catenin signaling is aberrantly activated in a variety of cancer including colorectal cancer, and plays a critical role in CSC maintenance. Here it is shown that a novel SCD inhibitor, DSP-0692, may inhibited Wnt/β-catenin signaling pathway through two different mechanisms in CRC. The effect of SCD inhibition on Wnt3A secretion, in which process post-translational fatty acylation (with palmitoleic acid) is required, was investigated. DSP-0692 led to reduced Wnt3A protein levels secreted in the media in Wnt3A-overexpressing cell line. This result was further confirmed by the TCF/LEF reporter assay in HCT116 incubated with conditional media from L-Wnt3A cells, with or without DSP-0692. DSP-0692 also inhibited exogenous Wnt3A-induced reporter activity. This indicates DSP-0692 may inhibit the β-catenin/Tcf transactivation by additional mechanism in addition to inhibiting Wnt secretion. To clarify this additional mechanism, the effect of DSP-0692 on nuclear translocation of β-catenin was investigated. SCD inhibition led to a decrease of β-catenin amounts within the nucleus. Currently, how SCD inhibition affects nuclear translocation of β-catenin is being investigated. It was confirmed that DSP-0692 exhibited lower IC50 value to sphere cells carrying mutations related to Wnt/β-catenin signaling pathway than those without these mutations. Next, the effect of SCD inhibition on Wnt/β-catenin signaling pathway in vivo was investigated. Treatment with DSP-0692 in a SW620 colorectal cancer xenograft model suppressed mRNA expression of the Wnt/β-catenin target genes, as well as CSC marker gene. Taken together, these results suggest fatty acid desaturation in tumor cells may have an impact on Wnt/β-catenin signaling pathway and involve in CSC maintenance. The approaches targeting SCD could be a new therapeutic strategy for anti-CSC therapy in CRC. Citation Format: Yudai Furuta, Yuichi Fukunaga, Satoshi Ikeda, Erina Koga, Hiroki Umehara, Tsuguteru Otsubo, Manabu Watanabe, Futoshi Hasegawa, Hitoshi Ban, Eiji Sugaru. Colorectal cancer cell lines harboring mutation in Wnt/β-catenin pathway shows enhanced sensitivity to the stearoyl-CoA desaturase (SCD) inhibitor, DSP-0692 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5480.

Raddeanin A inhibits growth and induces apoptosis in human colorectal cancer through downregulating the Wnt/β-catenin and NF-κB signaling pathway

AimsColorectal cancer (CRC) remains one of the most lethal human malignancies with high incidence and lack of effective therapy. Raddeanin A (RA), an active triterpenoid saponins, has been demonstrated the ability to inhibit the growth of tumor. But the therapeutic effects and mechanisms of RA in CRC remain elusive. Here, we investigated the efficacy and mechanism of RA in CRC both in vitro and in vivo. Main methodsCell viability was investigated to evaluate cytotoxic activity by MTT method. Apoptosis induced by RA was studied using Annexin V-FITC/PI binding and JC-1 staining by flow cytometry analysis. The xenograft mouse model of CRC was used to investigate anti-tumor effects in vivo. The key proteins involved in mitochondrial apoptotic, Wnt/β-catenin and NF-κB pathway were detected by Western blotting, Immunofluorescence, and Immunohistochemistry. Key findingsRA induced apoptosis and inhibited cell proliferation of SW480 and LOVO cells in a concentration-dependent manner. Moreover, RA efficiently inhibited tumor growth in xenograft mouse model. RA could down regulate the Wnt/β-catenin signaling to display anti-tumor effects via suppression of p-LRP6, induction of AKT inactivation, removal of GSK-3β inhibition and attenuation of β-catenin. Meanwhile, RA also suppressed the NF-κB pathway by decreasing the phosphorylation of IKBα to induce subsequently mitochondrial apoptotic pathway. SignificanceIn summary, RA suppressed the growth and triggered the apoptosis of CRC through discontinuing Wnt/β-catenin signaling and inhibiting the NF-κB pathway. These findings suggested that RA may hold a promise as a novel therapeutic agent for CRC therapy.

PO-143 Frequent loss of annexin A1 expression in head and neck squamous cell carcinomas by transcriptional regulatory mechanisms

IntroductionColorectal cancer (CRC) is amongst most frequent tumour types worldwide. Survivin, the smallest member of the inhibitors of apoptosis (IAP) family, is a well-established resistance marker, highly expressed in all tumour types including CRC. The aim of our study was to ascertain whether cellular localization of Survivin might affect the response of CRC cells to the first-line treatment with the topoisomerase I inhibitor irinotecan (IT).Material and methodsThe methods used were DNA cloning, site-directed mutagenesis, transfection and generation of stable clones, immune-fluorescence, colony-forming assays, flow cytometry, ELISA, PCR-based homologous recombination (HR) repair activity assays, western blot analysis, γH2AX foci formation and chromosomal aberration assays.Results and discussionsIn order to examine the effects of Survivin expression and localization on the response to IT, we transfected the p53-proficient CRC cell line (SW48) with the recombinant vector expressing Survivin-GFP fusion protein mainly localised to the cytoplasm. We also generated stable SW48 clones mutated in the nuclear export sequence (NES) of Survivin leading to its retention and accumulation in the nucleus. The proper localization of the fusion protein variants (∼42 kDa) i.e. the functional Survivin-GFP nuclear shuttling by passive diffusion and the active nuclear export were proven by leptomycin B (nuclear export inhibitor), leading to accumulation of the fusion protein in the nucleus. In colony-forming assays the striking difference between the clones was observed: the Survivin-NES-GFP clone was hypersensitive towards IT as compared to the Survivin-GFP clone or parental cells. Flow-cytometric cell-cycle distribution analyses showed that IT induced a prolonged G2-arrest (without induction of senescence) and a higher apoptotic frequency in the Survivin-NES clone, as compared to the parental cell line and the Survivin-GFP clone. Most interesting, the NES clone showed a low repair efficiency of DNA double-strand breaks (DSBs) by HR and lots of aberrant mitoses with a high clastogenic frequency, underlying the onset of mitotic catastrophe.ConclusionCytoplasm-localised Survivin with a normal shuttling function enhanced reproductive survival upon IT exposure and showed a reduced amount of IT-induced DSBs, probably supporting the repair by HR, while the export-impaired NES-Survivin led to an extremely toxic response towards IT. Therefore, Survivin localization could be a prognostic marker for topoisomerase I inhibitor-based CRC therapy.

Abstract LB-089: Targeting WNT signaling in vivovia Tankyrase inhibition

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States. Approximately 95% of CRCs show hyperactive WNT signaling, yet there are no targeted therapies that effectively suppress the WNT pathway. The majority (~80%) of these tumors harbor a mutation in APC, a scaffold component of the ‘destruction complex', which is a critical negative regulator of WNT. Using genetically defined mouse models of CRC, we recently showed that restoring Apc function can induce WNT suppression, and rapid and sustained disease regression. While there is no way to restore normal APC protein in human CRCs, it may be possible to restore the function of the APC-associated destruction complex by inhibiting Tankyrase (TNKS) enzymes. Tankyrase inhibitors have been shown to stabilize AXIN, prevent degradation of the destruction complex, and decrease levels of WNT signaling. While in vitro studies have shown promise, it is unclear whether TNKS inhibition could provide a therapeutic benefit in vivo. Some published work reports significant anti-tumor activity, while others suggest overt dose-limiting toxicity. Thus, while TNKS inhibition holds significant potential, it is not clear whether TNKS enzymes are a viable target for CRC therapy. To circumvent the limitations of drug delivery, potency, and specificity, we generated mice with inducible, fluorescent reporter-linked shRNAs targeting Tnks and its redundant family member Tnks2. Here we show that potent, simultaneous shRNA-mediated knockdown of both genes is sufficient to stabilize Axin in vivo, similar to treatment with a pan-TNKS inhibitor, XAV939. We show that Tnks1/2 knockdown stabilizes Axin, suppresses WNT signaling, and blocks hyperproliferation driven by Apc loss. In contrast to current studies using small molecule TNKS inhibitors, our work suggests that there is a viable therapeutic window of TNKS1/2 inhibition in vivo, and that this strategy enables control of hyperactive WNT signaling in CRC and other WNT-associated cancers. Citation Format: Emma M. Schatoff, Benjamin I. Leach, Miguel Foronda, Maria Paz Zafra, Sukanya Goswami, Lukas E. Dow. Targeting WNT signaling in vivovia Tankyrase inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-089.

The expression of telomere-related proteins and DNA damage response and their association with telomere length in colorectal cancer in Saudi patients.

BackgroundColorectal cancer is the leading cause of cancer-related deaths in Saudi Arabia. Cancer has a multifactorial nature and can be described as a disease of altered gene expression. The profiling of gene expression has been used to identify cancer subtypes and to predict patients’ responsiveness. Telomere-associated proteins that regulate telomere biology are essential molecules in cancer development. Thus, the present study examined their contributions to colorectal cancer progression in Saudi patients.MethodsThe expression of hTERT, TRF1, TRF2, POT1, ATR, ATM, Chk1 and Chk2 were measured via real-time PCR in matched cancerous and adjacent tissues of CRC patients. The protein level of hTERT, TRF1, TRF2, ATR, ATM, Chk1 and Chk2 were measured using immunohistochemistry. A region of hTERT core promoter was sequenced via Sanger sequencing. Methylation of CTCF binding site was examined via methylation-specific PCR. Finally, the length of telomere was estimated using q-PCR.ResultsOur results showed that POT1, ATR, Chk1 and Chk2 show increased expression in CRC relative to the adjacent mucosa. The expression levels of each gene were associated with clinicopathological characteristics of patients with CRC. There was a positive correlation between the age of the patients and hTERT expression. Regarding tumor site, telomere length, ATR, ATM and Chk1 were shown to be altered. No somatic mutation was detected in hTERT core promoter, and no differences in methylation patterns at CTCF binding site in the promoter between normal and cancer tissues.ConclusionAnalysis of targeted genes expression in colorectal cancer based on the clinical variables revealed that tumor location and age could have a role in gene expression and telomere length variations and this could be taken under consideration during CRC diagnosis and therapy. Other epigenetic mechanisms could influence hTERT expression in cancers. Our findings warrant further validation through experiments involving a larger number of patients.

Abstract B32: Targeting WNT signaling in vivo via Tankyrase inhibition

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States. Approximately 95% of CRCs show hyperactive WNT signaling, yet there are no targeted therapies that effectively suppress the WNT pathway. The majority (~80%) of these tumors harbor a mutation in APC, a scaffold component of the “destruction complex,” which is a critical negative regulator of WNT. Using genetically defined mouse models of CRC, we recently showed that restoring Apc function can induce WNT suppression and rapid and sustained disease regression. While there is no way to restore normal APC protein in human CRCs, it may be possible to restore the function of the APC-associated destruction complex by inhibiting Tankyrase (TNKS) enzymes. Tankyrase inhibitors have been shown to stabilize AXIN, prevent degradation of the destruction complex, and decrease levels of WNT signaling. While in vitro studies have shown promise, it is unclear whether TNKS inhibition could provide a therapeutic benefit in vivo. Some published work reports significant antitumor activity, while others suggest overt dose-limiting toxicity. Thus, while TNKS inhibition holds significant potential, it is not clear whether TNKS enzymes are a viable target for CRC therapy. To circumvent the limitations of drug delivery, potency, and specificity, we generated mice with inducible, fluorescent reporter-linked shRNAs targeting Tnks and its redundant family member Tnks2. Here we show that potent, simultaneous shRNA-mediated knockdown of both genes is sufficient to stabilize Axin in vivo, similar to treatment with a pan-TNKS inhibitor, XAV939. We show that Tnks1/2 knockdown stabilizes Axin and suppresses WNT signaling, but surprisingly, does not disrupt intestinal architecture. Importantly, Tnks1/2 silencing blocks hyperproliferation and tumor development in mice following Apc disruption. In contrast to current studies using small-molecule TNKS inhibitors, our work suggests that there is a viable therapeutic window of TNKS1/2 inhibition in vivo, and that this strategy enables control of hyperactive WNT signaling in CRC and other WNT-associated cancers. Citation Format: Emma M. Schatoff, Benjamin I. Leach, Miguel Foronda, Maria Paz Zafra, Lukas Dow. Targeting WNT signaling in vivo via Tankyrase inhibition [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B32.

Abstract 4836: A colorectal cancer organoid library provides a promising screening tool for assessment of chemosensitivity

Abstract Colorectal cancer (CRC) is a leading cause of cancer mortality and the third most common cancer worldwide. Annually, there are more than 900,000 cases occur worldwide, nearly 500,000 of which are fatal. CRC have accumulated defects in oncogenes and tumor suppressor genes. Many CRCs likely arise through clonal outgrowth and evolution of an epithelial cell or cells in an adenomatous precursor lesion via so-called adenoma-carcinoma sequence. Intensive studies have uncovered that the spectra of these genetic modifications are highly variable across the tumors. While genetic changes in tumors of individual patients can be assessed in several stratified methods, these data still need to be properly interpreted for necessitating model systems for analysis of chemosensitivity of CRC patients, which could enable us to organically coordinate among genotype-to-phenotype correlations, clinicopathological features and clinical outcomes. One of the most plausible models for implementing this magnificent scheme is most likely to be “organoid”, which is a novel 3D intestinal stem cell culture system and has successfully reconstitute normal and cancerous colonic crypts in in vitro through using the specific niche factors in a dish, such as EGF, Wnt3a, Rspondin-1 and Noggin, together with a TGF-b inhibitor and a p38 inhibitor. Two groups, Van der Wetering et.al and Fujii et.al, have previously reported the establishment of organoid biobank of colorectal cancer patients, and both of them proved the usefulness of this platform for underpinning the significance of genotype-phenotype analysis in every single patient and providing insights into tailor-made therapy in CRC. In this present study, we generated a pairs of cancerous and normal adjacent organoids from a number of CRC patients and aimed to figure out sustainability and reproducibility of CRC organoids especially in terms of phenotypic and genotypic features even after several times of passage. We compared growth activity and histopathological features of the CRC organoids among several time points after the passage by evaluating ki-67 labeling index and morphology of the organoids. In order to confirm the stability of genetic status of representative cancer-related genes, we focused on TP53, KRAS, BRAF, and MSI status. We then tried to elucidate the difference of chemosensitivity between MSI-high and MSI-low/MSS CRC cases with similar genetic background in most of the representative cancer-related genes. Although further in-depth studies on CRC organoids are needed, our approach would be instrumental in accurately predicting the drug sensitivity of CRC patients, which could possibly lead to the establishment of personalized medicine in CRC. Citation Format: Naoya Sakamoto, Akira Ishikawa, Ririno Honma, Takao Hinoi, Naohide Oue, Kazuhiro Sentani, Shoichiro Mukai, Tomohiro Adachi, Hiroyuki Egi, Hideki Ohdan, Wataru Yasui. A colorectal cancer organoid library provides a promising screening tool for assessment of chemosensitivity [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 4836. doi:10.1158/1538-7445.AM2017-4836

LASP2 suppresses colorectal cancer progression through JNK/p38 MAPK pathway meditated epithelial-mesenchymal transition

BackgroundLASP2 (LIM and SH3 Protein 2) is a small focal adhesion protein belongs to nebulin protein family. As the newest member of nebulette family, the function of LASP2 remains to be identified.MethodsThe relationship between LASP2 expression and clinical characteristics of CRC was analyzed in 89 paraffin-embedded archived CRC specimens by immunohistochemistry (IHC). The effects of LASP2 on cell growth and migration were examined in vitro, using CCK-8 and transwell assays. Western blotting was performed to examine the impact of LASP2 on the SAPK/JNK and MAPK signaling pathways.ResultsIn the present study, we observed a decreased LASP2 expression in clinical colorectal cancer samples compared with paired normal tissues. A negative correlation was also found between LASP2 and poor prognosis of CRC patients. Gain- and loss-of-function approaches revealed that LASP2 plays inhibitory effects on the growth and migration of human CRC cells in vitro. Western-blot results showed that LASP2 could attenuate epithelial-mesenchymal transition (EMT) to accomplish its suppression on CRC aggression. In LASP2 knocked down CRC cells, EMT was inhibited along with the inactivation of JNK/p38 MAPK pathway. Consistently, treatment of JNK inhibitor (JNK inhibitor II) together with p38 inhibitor (SB203580) could resume the process of EMT. Interestingly, we found a negative relationship between LASP2 and LASP1 expression in both CRC cell lines and tumors tissues, which suggests their converse function in CRC progression.ConclusionsAll the findings indicated that LASP2 may play a significant role in suppressing CRC progression and provided a novel biomarker for CRC therapy.

The therapeutic potential of targeting the BRAF mutation in patients with colorectal cancer.

Colorectal cancer is among the most lethal malignancies globally. BRAF is a member of the RAS/RAF/MEK/ERK signaling pathway. Its constitutive activation can result in increased cellular growth, development, invasion, and resistance to therapy. A mutation of the BRAF gene is present in 5-10% of metastatic colorectal cancers. BRAF mutations have been found to predict a lack of benefit to anti-EGFR therapy in metastatic CRC. Furthermore, CRC containing the BRAF V600E mutation display an innate resistance to BRAF inhibitors. The mechanisms of cell resistance can be explained at least in part by ERK dependent and ERK in-dependent pathway. Clinical trials evaluating the combinations of BRAF, PI3K, EGFR, and/or MEK inhibitors have revealed promising activity in BRAF mutant containing CRCs. There may be some benefit from future studies that focus on improving the efficacy of combined therapy in CRC with respect to the sustained effects. The aim of current review is to give an overview about the current status and prospective regarding the therapeutic potential of targeting BRAF mutant colorectal cancer.

A systematic review of the performance of the SEPT9 gene methylation assay in colorectal cancer screening, monitoring, diagnosis and prognosis.

The applications of the SEPT9 assay are expanding from CRC early diagnosis to screening, therapeutic effect monitoring and prognosis prediction. Its performance in these areas has not been thoroughly examined. We aim to evaluate the performance of the SEPT9 assay in CRC screening, diagnosis and therapy by reviewing the current data published in these aspects. The Ovid MEDLINE, EMBASE, CBMdisc (China Biology Medicine disc) and CJFD (Chinese Journal Full - text Database) database were searched for potential reports on the assay performance. Letters, reviews, meta-analysis and guidelines, basic research studies and articles irrelevant to mSEPT9 detection assays were excluded. Finally, data from 19 studies was summarized and systematically reviewed to clarify the assay performance. 2/3 algorithm provided the best overall performance in diagnosis and screening, while the 1/3 algorithm exhibited the best sensitivity in screening. The combination of SEPT9 assay with FIT and/or CEA enhanced the CRC detection rate in screening. The SEPT9 assay appeared to be effective in monitoring the therapeutic effect and may potentially predict the CRC recurrence and survival. The SEPT9 assay exhibited satisfactory performance in CRC diagnosis and screening, while more evidence is needed for therapeutic effect monitoring and prognosis prediction.

Modified nanoparticle mediated IL-12 immunogene therapy for colon cancer

For the past few years, immunotherapy has recently shown considerable clinical benefit in CRC therapy, and the application of immunologic therapies in cancer treatments continues to increase perennially. Interleukin-12, an ideal candidate for tumor immunotherapy, could activate both innate and adaptive immunities. In this study, we developed a novel gene delivery system with a self-assembly method by MPEG-PLA and DOTAP(DMP) with zeta-potential value of 38.5mV and size of 37.5nm. The supernatant of lymphocytes treated with supernatant from Ct26 transfected pIL12 with DMP could inhibit Ct26 cells growth ex vivo. Treatment of tumor-bearing mice with DMP-pIL12 complex has significantly inhibited tumor growth at both the subcutaneous and peritoneal model in vivo by inhibiting angiogenesis, promoting apoptosis and reducing proliferation. The IL-12 plasmid and DMP complex may be used to treat the colorectal cancer in clinical as a new drug.

Screening of 129 FDA approved anti-cancer drugs in colorectal cancer cell lines resistant to oxaliplatin or irinotecan (SN38).

642 Background: In metastatic colorectal cancer (mCRC) only 3 cytotoxic drugs (oxaliplatin, irinotecan and fluorouracil (5-FU)) are approved and the first and second line response rates are about 50% and 10-15%, respectively. Thus, new treatment options are needed. Novel anti-cancer drug candidates are primarily tested in an environment of drug resistance and the majority of novel drug candidates fail during clinical development. Therefore, “repurposing” of drugs has emerged as a promising strategy to apply established drugs in novel indications. The aim of this project was to screen established anti-cancer drugs to identify candidates for testing in mCRC patients relapsing on standard therapy. Methods: We applied 3 parental (drug sensitive) CRC cell lines (HCT116, HT29 and LoVo) and for each cell line also an oxaliplatin and irinotecan (SN38) resistant cell line. We obtained 129 FDA approved anti-cancer drugs from the Developmental Therapeutics Program (DTP) at the National Cancer Institute (NCI) ( https://dtp.cancer.gov/ ). The parental HT29 cell line and the drug resistant sublines HT29-SN38 and HT29-OXPT were exposed to 3 concentrations of each of the anti-cancer drugs. The effect on cell viability was analyzed by MTT assays. Nine of the drugs were analyzed for effect in the LoVo and HCT116 and the SN38- and oxaliplatin-resistant derived cell lines. Results: None of the drugs caused evident differential response between the resistant and sensitive cells or between the SN38 and oxaliplatin resistant cells. The screening confirmed the resistance as the cells displayed resistance to drugs in the same class as the one they were made resistant to. Of the drugs, 45 decreased cell viability in the HT29 parental and oxaliplatin- or SN-38 resistant cell lines. Nine drugs were tested in all nine CRC cell lines and eight decrease cell viability in the nine cell lines. These included drugs in different classes such as epigenetic drugs, antibiotics, mitotic inhibitors and targeted therapies. Conclusions: This study revealed several possible new “repurposing” drugs for CRC therapy, by showing that 45 FDA-approved anti-cancer drugs decrease cell viability in CRC cell lines with acquired drug resistance.

Spectrum of Gene Mutations in Colorectal Cancer: Implications for Treatment.

Gene mutations acquired during colorectal carcinogenesis remain drivers of cancer progression in the metastatic setting. KRAS and NRAS mutations define a population refractory to anti-epidermal growth factor receptor (EGFR) antibodies, either as single agents or in combination with standard chemotherapy. High-sensitivity extended RAS testing is currently a requirement to select anti-EGFR therapy irrespective of treatment line, thus limiting unnecessary exposure and expense in patients unlikely to respond. Multiple genetic alterations driving resistance to anti-EGFR monoclonal antibodies have been described, with significant overlap in primary and acquired resistance mechanisms, in line with a clonal selection process. Some of them have been validated as targets for therapeutic intervention in clinical trials, such as ERBB2 amplifications. With advances in drug development and better understanding of the dynamics of target inhibition, additional gene alterations are now promising positive predictive markers for matched targeted therapies in CRC, including BRAF V600E and RNF43 mutations. Furthermore, the microsatellite instable hypermutated colorectal cancer population is particularly sensitive to immune checkpoint inhibitors. In this article, we review the expanding landscape of druggable gene alterations in metastatic colorectal cancer.

Comprehensive genomic profiling of advanced colorectal carcinoma in the course of clinical care to identify KRAS insertions not detected by focused molecular testing.

497 Background: As activating RAS mutations have been shown to predict lack of benefit from anti-EGFR therapies in advanced CRC, NCCN guidelines recommend testing for KRAS exon 2 and non-exon 2 mutations; however, these alterations are thought to explain only a subset of de novo resistance to targeted therapy. In a large set of CRC assayed with comprehensive genomic profiling (CGP) in the course of clinical care, we assessed the frequency of less common KRAS short insertions that may predict failure of anti-EGFR therapy. Methods: 4,422 CRC cases were assayed with CGP performed on hybridization-captured, adaptor ligation based libraries to a mean coverage depth of > 650X for at least 236 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer. All classes of genomic alterations (GA) were identified, including base pair substitutions, insertions/deletions, copy number alterations, and rearrangements. Pertinent available prior molecular testing results and clinical history was reviewed for selected cases. Results: Out of 4,422 CRC cases analyzed, KRAS short variants were identified in 50.9% of cases. A majority of cases contained a KRAS alteration at either codon G12 (35.5%) or G13 (9.1%), while alterations at codons Q61 or A146 were identified in 2.2% and 3.1% of cases, respectively. KRAS insertions were identified in 8 ( < 0.5%) cases. All KRAS insertions identified fell within codons 9-13, and 6/8 cases harbored V9_G10 insertions. Out of 6 patients with prior KRAS testing results available, 5 (83%) were negative by previous testing. Conclusions: CGP identifies KRAS insertions within or adjacent to hotspot regions in CRC cases which have previously tested negative for KRAS mutations. Given the importance of KRAS alterations in predicting lack of response to anti-EGFR therapies in CRC, accurate detection of these alterations in the course of clinical care is essential for effective treatment. CGP offers the possibility of identifying KRAS insertions that may impact efficacy of anti-EGFR targeted therapy and should be considered when previous focused testing for KRAS mutations is negative.

Abstract LB-119: MGMT immunohistochemistry (IHC) improves patients’ selection for temozolomide (TMZ) treatment in advanced chemorefractory MGMT-methylated colorectal cancer

Abstract Background: We showed that standard dose TMZ (150 mg/mq/day day1-5q28) is tolerable and active in 32 heavily pre-treated patients with advanced CRC and MGMT promoter methylation (Pietrantonio et al., Ann Oncol 2014). In a subsequent study, we included 32 pts treated with dose-dense TMZ (75 mg/mq/day day 1-21q28, for up to 6 cycles or until progression/unacceptable toxicity). Additional predictive markers are needed for improved selection of patients for TMZ therapy in metastatic CRC. Retained MGMT expression by IHC was proposed as marker for negative selection of pts with brain tumors. We conducted a retrospective analysis to assess MGMT expression in our 2 studies and to correlate this with the outcomes. Methods: All 64 patients included in the 2 studies were defined as MGMT methylation-positive according to methylation-specific PCR. A total of 40 patients had tissue available for IHC. Expression of nuclear MGMT protein was defined scored semiquantitatively according to extension and intensity. Using ROC analysis, IHC score <4 was considered as MGMT-low expression vs. IHC score 4-12 as MGMT-high. Extended RAS-BRAF mutations were assessed by Sanger sequencing. Results: Herein, we report for the first time the results of the dose-dense TMZ study. We obtained 4 confirmed partial responses and 2 stable disease, accounting for a response rate of 12% and a clinical benefit of 18%. Median PFS was 1.8 months. OS data are not mature. Thus, the results are comparable to those obtained in the previous study. MGMT-low expression was found in 15 (38%) samples and MGMT-high in 25 (62%). RAS-BRAF mutations were found in 28 (70%) pts and were not correlated with MGMT expression (p = 1). Response rate was significantly higher in patients with MGMT-low (53%) vs. those with MGMT-high (p<0.0001). Progression-free survival was significantly longer in MGMT-low vs. MGMT-high(5 vs. 2.3 months; p = 0.001). Further analyses are ongoing to validate MGMT IHC as a biomarker and to correlate it with quantitative gene methylation analysis. Conclusions: In this translational study on MGMT methylated CRC, absence of MGMT expression was significantly correlated with tumor response and benefit from TMZ treatment. Citation Format: Filippo De Braud, Filippo Pietrantonio, Maria Di Bartolomeo, Katia Fiorella Dotti, Claudia Maggi, Roberto Iacovelli, Marta Caporale, Rosa Berenato, Federica Perrone, Elena Tamborini, Alessio Pellegrinelli, Stefano Federici, Fabrizio Festinese, Giuseppe Pelosi, Ilaria Bossi, Paola Valentina Consonni, Susanna Maggi, Massimo Milione. MGMT immunohistochemistry (IHC) improves patients’ selection for temozolomide (TMZ) treatment in advanced chemorefractory MGMT-methylated colorectal cancer. [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 LB-119. doi:10.1158/1538-7445.AM2015-LB-119

In-vitro characterisation of a novel celecoxib microbead formulation for the treatment and prevention of colorectal cancer.

Colorectal cancer (CRC) is a life-threatening disease that can develop as a consequence of a sustained chronic inflammatory pathology of the colon. Although not devoid of side effects, the anti-inflammatory drug celecoxib (CLX) has been shown to exert protective effects in CRC therapy. The purpose of this study was to develop and characterise a novel CLX microbead formulation suitable for use in the treatment and prevention of CRC, which has the potential to minimise the side effects associated with CLX. The study involved the assessment of the effectiveness of CLX formulations in an in-vitro cell model (HT29 cells) and a comparison of these effects to that of the marketed CLX product, Celebrex. Liquid CLX formulations were developed as precursors to microbead formulations. The effect of liquid CLX formulations on HT29 cell viability (MTT and flow cytometry apoptotic assays) and motility (scratch wound assay) were assessed and compared with the effect of Celebrex. A correlation between the in-vitro dissolution performance of the formulations and the effect in the cell model was also explored. Liquid CLX formulations were translated into an optimised CLX microbead formulation, and a colonic targeted sustained release coat (Surelease) was applied to the beads with the aim of producing a formulation for a future in-vivo study to compare the effect of the coated CLX microbeads versus Celebrex in the attenuation of CRC tumours and inflammation in a CRC murine model. The production of CLX microbeads was scaled-up using vibrating-jet encapsulation technology to allow for the development of an optimised dissolution profile to enable colonic release. In-vitro cell viability and motility were shown to be significantly reduced after treatment with CLX liquid formulations relative to the control, whereas the results for treatment with Celebrex were comparable with the control. Dissolution experiments and correlation analysis demonstrated that the formulations that showed a greater extent of drug release had reduced cell viability and motility. The CLX liquid formulations were translated into colon-targeted CLX microbeads suitable for use in a future in-vivo mouse study. These results represent a significant step forward in the chemopreventative treatment of CRC using CLX, as the microbead formulation developed suggests the possibility of presenting CLX in a format that has the potential to minimise gastrointestinal and cardiovascular side effects.

Abstract 627: Immunogenic cell death as novel immune response mechanism to EGFR-targeted therapy in CRC

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide. The major improvement in the median overall survival of mCRC patients was mainly due to the introduction of new active chemotherapeutic and biomolecular targeting agents. Most chemotherapeutic agents and radiotherapy kill tumor cells through an apoptotic pathway, which is considered as a non-immunogenic. However, anthracyclines, oxaliplatin or ionizing irradiation are able to induce a potent immune response in vivo, when injected in immunocompetent mice. Dying cells are readily taken up by dendritic cells (DC) that then cross-present their associated antigens for effective T cell activation. Herein we propose that the treatment with an anti-EGFR antibody (Cetuximab) in combination with chemotherapy FOLFIRI (5-fluorouracile, leucovorin, irinotecan) induces a immunogenic cell death (ICD) modality, which contributes to its clinical efficacy in patients with mCRC expressing EGFR and the wild type (wt) form of the viral oncogene v-Ki ras2. Our results, obtained on CRC cell lines provide evidence, for the first time, that treatment with Cetuximab alone or in combination with a chemotherapeutic regimen (FOLFIRI) directs dying cells towards an ICD process characterized by phosphorylation of eIF2α and, most important, increased phagocytosis by DCs. However, not all cell lines wt for KRAS (expressing EGFR) displayed the same response. As expected, Cetuximab blocked the pathways leading to AKT and ERK phosphorylation. However, we also observed that Cetuximab, and to a lesser extent the F(ab)2 fragments of Cetuximab, was inducing some phosphorylation of the EGFR in KRAS wt responding cell line, which was lower than that induced by EGF . Hence Cetuximab is uncoupling the activation of the signaling pathway induced via EGFR from EGFR phosphorylation, suggesting an alternative activation of the receptor. This allowed us to postulate that Cetuximab-induced phagocytosis may be the effect of an alternative activation of the EGFR. Indeed, we could not detect any phagocytosis in cell lines where the phosphorylation of the receptor did not occur in response to Cetuximab. These results identify ICD as novel immune response mechanism to EGFR targeted therapy alone or in combination with chemotherapy and possibly genetic alterations of the EGFR-signalling pathway, could interfere with the induction of ICD. Citation Format: Giuseppe Penna, Chiara Pozzi, Elena Magni, Giuseppe Curigliano, Maria Giulia Zampino, Maria Rescigno. Immunogenic cell death as novel immune response mechanism to EGFR-targeted therapy in CRC. [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 627. doi:10.1158/1538-7445.AM2014-627

Abstract 4397: Therapeutic microRNA agaisnt KRAS mutant colorectal cancer

Abstract Background: The anti-epidermal growth factor receptor (EGFR) monoclonal antibodies are the molecular target drugs, dependent on v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations status in metastatic colorectal cancer (mCRC). Anti-EGFR monoclonal antibody based chemotherapy is the standard treatment for mCRC with KRAS wild-type. However, the issue of resistance to this therapy in CRC with KRAS mutations (approximately 50% of CRC) remains unresolved. The aim of this study is to identify microRNAs (miRNAs) that govern the resistance to anti-EGFR monoclonal antibody in CRC with KRAS mutations. Experimental Design: We first established the KRAS mutant cell model using HEK293 and MRC5 cells by introducing KRASG12V plasmid. The miRNA expression profiles were compared between these cells using miRNA microarray analysis. We selected the candidate a miRNA which down-regulates the downstream signal of KRAS using luciferase reporter assays. Assessment of target genes of the candidate a miRNA was performed in vitro. Next, anti-tumor effects by supplement of a miRNA were evaluated in vitro and in vivo. Results: We picked up miRNAs, of which expressions decreased in both HEK293 and MRC5 cells by introduction of KRASG12V plasmid in miRNA array analysis. Among them, administration of mature-miR-X suppressed SRE and AP1 activity in KRAS mutant CRC cell lines such as DLD1G13D, SW480 G12V, and HCT116 G13D using dual luciferase reporter assay. Furthermore, miR-X regulated not only Ras/Raf/MAPK pathway but also suppressed PI3K/Akt pathway in vitro. Administration of mature-miR-X suppressed proliferation and induced apoptosis of the CRC cell lines with KRAS mutations in vitro. Systemic mature-miR-X administration into tail veins of nude mice significantly inhibited the growth of pre-established DLD1G13D xenografts. miR-X level was significantly lower in tumor tissues compared to normal mucosa. Conclusions: miR-X may overcome anti-EGFR therapy resistance in CRC with KRAS mutations by regulating Ras/Raf/MAPK pathway and PI3K/Akt pathway. Our data suggests that miR-X could be suitable for a novel therapy for mCRC patients with KRAS mutations. Citation Format: Masayuki Hiraki, Junichi Nishimura, Mamoru Uemura, Taishi Hata, Ichiro Takemasa, Tsunekazu Mizushima, Hirofumi Yamamoto, Yuichiro Doki, Masaki Mori. Therapeutic microRNA agaisnt KRAS mutant 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 4397. doi:10.1158/1538-7445.AM2014-4397