Microsatellite Instability Colorectal Cancer Cell Research Articles

Frameshift mutations in peripheral blood as a biomarker for surveillance of Lynch syndrome.

Lynch syndrome is a hereditary cancer predisposition syndrome caused by germline mutations in DNA mismatch repair genes, which lead to high microsatellite instability and frameshift mutations at coding mononucleotide repeats in the genome. Recurrent frameshift mutations in these regions are thought to play a central role in the increased risk of various cancers, but no biomarkers are currently available for the surveillance of high microsatellite instability-associated cancers. A frameshift mutation-based biomarker panel was developed and validated by targeted next-generation sequencing of supernatant DNA from cultured high microsatellite instability colorectal cancer cells. This panel supported selection of 122 frameshift mutation targets as potential biomarkers. This biomarker panel was then tested using matched tumor, adjacent normal tissue, and buffy coat samples (53 samples) and blood-derived cell-free DNA (cfDNA) (38 samples) obtained from 45 high microsatellite instability and mismatch repair-deficient patients. We also sequenced cfDNA from 84 healthy participants to assess background noise. Recurrent frameshift mutations at coding mononucleotide repeats were detectable not only in tumors but also in cfDNA from high microsatellite instability and mismatch repair-deficient patients, including a Lynch syndrome carrier, with a varying range of target detection (up to 85.2%), whereas they were virtually undetectable in healthy participants. Receiver operating characteristic curve analysis showed high sensitivity and specificity (area under the curve = 0.94) of the investigated panel. We demonstrated that frameshift mutations can be detected in cfDNA from high microsatellite instability and mismatch repair-deficient patients and asymptomatic carriers. The 122-target frameshift mutation panel described here has promise as a tool for improved surveillance of high microsatellite instability and mismatch repair-deficient patients, with the potential to reduce the frequency of invasive screening methods for this high-cancer-risk cohort.

Cold atmospheric plasma stabilizes mismatch repair for effective, uniform treatment of diverse colorectal cancer cell types

Mismatch Repair (MMR) mechanisms play a pivotal role in rectifying DNA replication errors and maintaining the stability of DNA microsatellite structure. Colorectal cancer (CRC) can be characterized into microsatellite stability (MSS) and microsatellite instability (MSI) subtypes based on the functionality of MMR. MSI CRC notably exhibits enhanced chemotherapy resistance, attributable to diminished MMR-related protein expression. Cold atmospheric plasma (CAP) has emerged as a promising treatment modality, demonstrating efficacy in inducing apoptosis in various cancer cells. However, the therapeutic impact of CAP on MSI colorectal cancer, and the underlying mechanisms remain elusive. In this study, we investigated the effects of CAP on MSI (MC38, HCT116, and LOVO) and MSS (CT26 and HT29) CRC cell lines. We are probing into the products of CAP treatment. Our findings indicate that CAP treatment induces comparable effects on apoptosis, reactive oxygen species (ROS), and reactive nitrogen species (RNS), as well as the expression of apoptosis-related proteins in both MSI and MSS cells. Mechanistically, CAP treatment led to an elevation in the expression of mismatch repair proteins (MLH1 and MSH2), particularly in MSI cells, which notably have been proven to facilitate the activation of apoptosis-related proteins. Collectively, our study reveals that CAP enhances apoptotic signaling and induces apoptosis in MSI colorectal cancer cells by upregulating the expression of MMR-related proteins, thereby reinforcing MMR stabilization.

Synthetical lethality of Werner helicase and mismatch repair deficiency is mediated by p53 and PUMA in colon cancer

Synthetic lethality is a powerful approach for targeting oncogenic drivers in cancer. Recent studies revealed that cancer cells with microsatellite instability (MSI) require Werner (WRN) helicase for survival; however, the underlying mechanism remains unclear. In this study, we found that WRN depletion strongly induced p53 and its downstream apoptotic target PUMA in MSI colorectal cancer (CRC) cells. p53 or PUMA deletion abolished apoptosis induced by WRN depletion in MSI CRC cells. Importantly, correction of MSI abrogated the activation of p53/PUMA and cell killing, while induction of MSI led to sensitivity in isogenic CRC cells. Rare p53-mutant MSI CRC cells are resistant to WRN depletion due to lack of PUMA induction, which could be restored by wildtype (WT) p53 knock in or reconstitution. WRN depletion or treatment with the RecQ helicase inhibitor ML216 suppressed in vitro and in vivo growth of MSI CRCs in a p53/PUMA-dependent manner. ML216 treatment was efficacious in MSI CRC patient-derived xenografts. Interestingly, p53 gene remains WT in the majority of MSI CRCs. These results indicate a critical role of p53/PUMA-mediated apoptosis in the vulnerability of MSI CRCs to WRN loss, and support WRN as a promising therapeutic target in p53-WT MSI CRCs.

(Phospho)proteomic Profiling of Microsatellite Unstable CRC Cells Reveals Alterations in Nuclear Signaling and Cholesterol Metabolism Caused by Frameshift Mutation of NMD Regulator UPF3A.

DNA mismatch repair-deficient colorectal cancers (CRCs) accumulate numerous frameshift mutations at repetitive sequences recognized as microsatellite instability (MSI). When coding mononucleotide repeats (cMNRs) are affected, tumors accumulate frameshift mutations and premature termination codons (PTC) potentially leading to truncated proteins. Nonsense-mediated RNA decay (NMD) can degrade PTC-containing transcripts and protect from such faulty proteins. As it also regulates normal transcripts and cellular physiology, we tested whether NMD genes themselves are targets of MSI frameshift mutations. A high frequency of cMNR frameshift mutations in the UPF3A gene was found in MSI CRC cell lines (67.7%), MSI colorectal adenomas (55%) and carcinomas (63%). In normal colonic crypts, UPF3A expression was restricted to single chromogranin A-positive cells. SILAC-based proteomic analysis of KM12 CRC cells revealed UPF3A-dependent down-regulation of several enzymes involved in cholesterol biosynthesis. Furthermore, reconstituted UPF3A expression caused alterations of 85 phosphosites in 52 phosphoproteins. Most of them (38/52, 73%) reside in nuclear phosphoproteins involved in regulation of gene expression and RNA splicing. Since UPF3A mutations can modulate the (phospho)proteomic signature and expression of enzymes involved in cholesterol metabolism in CRC cells, UPF3A may influence other processes than NMD and loss of UPF3A expression might provide a growth advantage to MSI CRC cells.

Epidermal growth factor receptor stabilizes programmed death ligand 1 by glycosylation in colorectal cancer with microstatellite instability status

Abstract Programmed death ligand-1 (PD-L1) is involved in inhibiting of T lymphocyte proliferation, producing cytokine, cytolytic activity, and suppressing of the immune response. Genes with molecular alterations involved in DNA mismatch repair promote cancer initiation and tumor progression. Clinical studies show that colorectal cancer (CRC) patients harboring microsatellite instability (MSI) have a higher anti-programmed cell death protein 1/PD-L1 immunotherapy response ratio compared with microsatellite stable subgroup patients. The underlying mechanism has however remained unclear. Here, we found that compared with microsatellite stable samples, PD-L1 was glycosylated and highly expressed both in MSI CRC cell lines and tissue samples. Specifically, PD-L1 was N-glycosylated at its N35, N192, N200, and N219 sites, and the four glycosylation sites were all responsible for PD-L1 degradation. Additionally, non-glycosylated PD-L1 underwent rapid degradation compared with glycosylated PD-L1 through the 26S proteasome pathway. The faster degradation of the non-glycosylated PD-L1 was ascribed to its binding to glycogen synthase kinase 3β via ubiquitination. This degradation phenotype was, however, not observed for glycosylated PD-L1. Significantly, glycosylated PD-L1 was up-regulated by activated epidermal growth factor receptor in MSI CRC cells. Together, our results indicate that epidermal growth factor receptor stabilized PD-L1 via glycosylation in MSI CRC cells, uncovering a novel role of PD-L1 in MSI CRC immunosuppression and disease progression. The study was approved by the Clinical Ethics Review Committee at the Six Affiliated Hospital of Sun Yat-sen University, China (Approval No. 2019ZSLYEC-005).

DNA methylation-mediated repression of miR-181a/135a/302c expression promotes the microsatellite-unstable colorectal cancer development and 5-FU resistance via targeting PLAG1

Microsatellite instability (MSI) defines a subtype of colorectal cancer (CRC) with typical clinicopathologic characteristics. CRCs with MSI (MSI CRCs) frequently acquire accelerated carcinogenesis and 5-FU resistance, and the exact underlying mechanism remains incompletely understood. Our previous study has identified the microRNA (miRNA) expression profile in MSI CRCs. In this study, three miRNAs (miR-181a, miR-135a and miR-302c) were validated by qRT-PCR to be dramatically decreased in 67 CRC samples. Proliferation and apoptosis assays demonstrated that miR-181a/135a/302c function as tumor suppressors via repressing PLAG1/IGF2 signaling. Moreover, we presented compelling evidence that restoration of miR-181a/135a/302c expression promoted sensitivity of MSI CRC cells to 5-FU treatment. miR-181a/135a/302c exerted their effect on chemoresistance through attenuating PLAG1 expression. Notably, the hypermethylation status of MSI CRC accounts for the decrements of miR-181a/135a/302c. Our results contribute to a better understanding of the mechanism of chemoresistance in MSI CRCs, and provide a clue for digging the biomarkers and therapeutic targets for CRC patients.

Elevated microRNA-23a Expression Enhances the Chemoresistance of Colorectal Cancer Cells with Microsatellite Instability to 5-Fluorouracil by Directly Targeting <i>ABCF1</i>

Colorectal cancer (CRC) is one of the most common tumor types worldwide. A frequent subtype of CRC is defined by a deficiency in the mismatch repair (MMR) pathway, constantly found in combination with microsatellite instability (MSI), which not only contributes to the pathogenesis of a large proportion of CRC, but also controls the response to multiple drugs used to treat CRCs. The most commonly used chemotherapeutic agent for CRC is 5-fluorouracil (5-FU). However, CRC with MSI frequently acquires 5-FU resistance, and the exact mechanism underlying how CRC cells acquire chemoresistance to 5-FU remains incompletely understood. Recently, emerging evidence has demonstrated that microRNAs (miRNAs) are key players in multidrug resistance. In this study, we aimed to characterize the expression profiles and functions of miRNAs in 5-FU-resistant CRC with MSI. We found that miR-23a was significantly elevated in MSI CRC cells and tissues compared to microsatellite stability (MSS) CRC cells and tissues. Ectopic expression of miR-23a increased the viability and survival of MSS CRC cells. Inversely, downregulation of miR-23a reduced viability in and promoted cell apoptosis in MSI CRC cells treated with 5-FU. Moreover, we demonstrated that ABCF1 is a direct target of miR-23a. Additionally, the expression of miR-23a was inversely correlated with the expression of ABCF1 in CRC tissues. Interestingly, repressing ABCF1 expression by either miR-23a overexpression or siABCF1 led to recovery of 5-FU sensitivity in MSI CRC cells. These data demonstrated that miR-23a enhances 5-FU resistance in MSI CRC cells through targeting ABCF1 and thus provided important implications for therapeutic approaches aiming to overcome MSI CRC resistance to 5-FU.

Methylation-induced loss of miR-484 in microsatellite-unstable colorectal cancer promotes both viability and IL-8 production via CD137L.

Colorectal cancer (CRC) exhibiting MSI (microsatellite instability) represents a well-defined subtype characterized by a deficient mismatch repair pathway and typical clinico-pathological features. Our objective was to identify the entire miRNome and its molecular pathological roles in MSI CRCs. We profiled miRNA expression in MSI CRCs and compared it with MSS counterparts. Microarray and qRT-PCR analysis identified eight miRNAs that could distinguish the MSI status of CRCs. MiR-484 was the most significantly decreased miRNA in MSI CRCs, primarily mediated by the CpG island methylator phenotype. MiR-484 functions as a tumour suppressor to inhibit MSI CRC cell viability in vitro and in vivo. Moreover, miR-484 repressed CD137L expression and thereby attenuated IL-8 production by MSI CRC cells. Our results contribute to a better understanding of the roles of dysregulated miRNAs in the distinct phenotypic features of MSI CRCs and indicate an option for early diagnosis and gene therapy for these patients.

Treatment with the PARP inhibitor, niraparib, sensitizes colorectal cancer cell lines to irinotecan regardless of MSI/MSS status.

BackgroundCells with homologous recombination (HR) deficiency, most notably caused by mutations in the BRCA1 or BRCA2 genes, are sensitive to PARP inhibition. Microsatellite instability (MSI) accounts for 10-15% of colorectal cancer (CRC) and is hypothesized to lead to HR defects due to altered expression of Mre11, a protein required for double strand break (DSB) repair. Indeed, others have reported that PARP inhibition is efficacious in MSI CRC.MethodsHere we examine the response to niraparib, a potent PARP-1/PARP-2 inhibitor currently under clinical evaluation, in MSI versus microsatellite stable (MSS) CRC cell lines in vitro and in vivo. We compiled a large panel of MSI and MSS CRC cell lines and evaluated the anti-proliferative activity of niraparib. In addition to testing single agent cytotoxic activity of niraparib, we also tested irinotecan (or SN-38, the active metabolite of irinotecan) activity alone and in combination with niraparib in vitro and in vivo.ResultsIn contrast to earlier reports, MSI CRC cell lines were not more sensitive to niraparib than MSS CRC cell lines¸ suggesting that the MSI phenotype does not sensitize CRC cell lines to PARP inhibition. Moreover, even the most sensitive MSI cell lines had niraparib EC50s greater than 10 fold higher than BRCA-deficient cell lines. However, MSI lines were more sensitive to SN-38 than MSS lines, consistent with previous findings. We have also demonstrated that combination of niraparib and irinotecan was more efficacious than either agent alone in both MSI and MSS cell lines both in vitro and in vivo, and that niraparib potentiates the effect of irinotecan regardless of MSI status.ConclusionsOur results support the clinical evaluation of this combination in all CRC patients, regardless of MSI status.

Abstract 3156: New candidate oncogenes discovered in microsatellite unstable colorectal cancer.

Abstract Around 15% of colorectal cancers (CRCs) show microsatellite instability (MSI). A characteristic high passenger mutation load has discouraged systematic mutation screens in MSI CRCs. To systematically search for novel MSI CRC oncogenes, the exomes of 25 MSI CRC-normal pairs were sequenced as the discovery set. Observed hot spots were confirmed by Sanger sequencing and further validated in a set of 86 MSI CRCs. Mutational hot spots were confirmed in 15 genes and three genes showed additional hot spot mutations in the validation set. These three sites were highly conserved across species. The hot spot sites of these three genes were screened in 75 microsatellite stable CRCs and 12 MSI CRC cell lines with negative results. Next, we analyzed the subcellular localization of wild-type and mutant proteins. The findings of this study support the idea that cancer genomes are heterogeneous and characterized by few, frequently mutated “mountains” (e.g. BRAF and KRAS) and numerous, less frequently mutated “hills”. The identified mutational hot spots may prove important in developing personalized tumor profiling and therapy. Citation Format: Alexandra E. Gylfe, Johanna Kondelin, Mikko Turunen, Heikki Ristolainen, Riku Katainen, Esa Pitkänen, Eevi Kaasinen, Ville Rantanen, Tomas Tanskanen, Heli J. Lehtonen, Kimmo Palin, Minna Taipale, Jussi Taipale, Laura Renkonen-Sinisalo, Heikki Järvinen, Jan Böhm, Jukka-Pekka Mecklin, Ari Ristimäki, Sari Tuupanen, Auli Karhu, Outi Kilpivaara, Pia Vahteristo, Lauri A. Aaltonen. New candidate oncogenes discovered in microsatellite unstable colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3156. doi:10.1158/1538-7445.AM2013-3156

Abstract 1085: TGFBR2-dependent alterations of glycosylation in MSI colorectal cancer

Abstract Aberrant glycosylation is a common feature of many malignancies including colorectal cancers. About 15% of colorectal cancers show the microsatellite instability (MSI) phenotype that is associated with a high frequency of biallelic frameshift mutations in the exon 3 coding mononucleotide microsatellite of the Transforming growth factor beta receptor 2 (TGFBR2) gene. If and how disruption of normal TGFBR2 signaling in these MSI colorectal cancer cells is linked to altered glycan pattern is largely unexplored. To address this issue we used the TGFBR2-deficient MSI colon carcinoma cell line HCT116 as a model system. Stable clones enabling doxycycline-inducible expression of wildtype TGFBR2 transgene were generated by recombinase-mediated cassette exchange (RMCE). In two independent clones dox-inducible expression of wildtype TGFBR2 protein and functional reconstitution of TGFBR2-mediated signal transduction as recognized by target gene regulation (SMAD7, SERPINE, c-myc) and Phospho-SMAD2 analysis was confirmed by Western blot and qRT-PCR analysis. Metabolic labeling experiments using the sialic acid precursor 3H-N-acetyl-mannosamine revealed a significant decline of 30% and hence newly synthesized sialoglycoproteins in TGFBR2 expressing cells. Therefore, our findings demonstrate that this model system can be used as a versatile tool to study TGFBR2-dependent effects. Furthermore, our data reveal a TGFBR2 gene-dependent aberrant glycosylation pattern in the MSI colon cell line HCT116. 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 1085. doi:1538-7445.AM2012-1085

Villin Expression Is Frequently Lost in Poorly Differentiated Colon Cancer

Colorectal cancers (CRCs) are classified as having microsatellite instability (MSI) or chromosomal instability (CIN); herein termed microsatellite stable (MSS). MSI colon cancers frequently display a poorly differentiated histology for which the molecular basis is not well understood. Gene expression and immunohistochemical profiling of MSS and MSI CRC cell lines and tumors revealed significant down-regulation of the intestinal-specific cytoskeletal protein villin in MSI colon cancer, with complete absence in 62% and 17% of MSI cell lines and tumors, respectively. Investigation of 577 CRCs linked loss of villin expression to poorly differentiated histology in MSI and MSS tumors. Furthermore, mislocalization of villin from the membrane was prognostic for poorer outcome in MSS patients. Loss of villin expression was not due to coding sequence mutations, epigenetic inactivation, or promoter mutation. Conversely, in transient transfection assays villin promoter activity reflected endogenous villin expression, suggesting transcriptional control. A screen of gut-specific transcription factors revealed a significant correlation between expression of villin and the homeobox transcription factor Cdx-1. Cdx-1 overexpression induced villin promoter activity, Cdx-1 knockdown down-regulated endogenous villin expression, and deletion of a key Cdx-binding site within the villin promoter attenuated promoter activity. Loss of Cdx-1 expression in CRC lines was associated with Cdx-1 promoter methylation. These findings demonstrate that loss of villin expression due to Cdx-1 loss is a feature of poorly differentiated CRCs.

Quercetin enhances 5-fluorouracil-induced apoptosis in MSI colorectal cancer cells through p53 modulation

Colorectal tumors (CRC) with microsatellite instability (MSI) show resistance to chemotherapy with 5-fluorouracil (5-FU), the most widely used pharmacological drug for CRC treatment. The aims of this study were to test the ability of quercetin (Q) and luteolin (L) to increase the sensitivity of MSI CRC cells to 5-FU and characterize the dependence of the effects on cells' p53 status. Two MSI human CRC-derived cell lines were used: CO115 wild type (wt) for p53 and HCT15 that harbors a p53 mutation. Apoptosis induction in these cells by 5-FU, Q and L alone, and in combinations was evaluated by TUNEL and western blot. The dependence of the effects on p53 was confirmed by small interference RNA (siRNA) in CO115 cells and in MSI HCT116 wt and p53 knockout cells. CO115 p53-wt cells are more sensitive to 5-FU than the p53-mutated HCT15. The combination treatment of 5-FU with L and Q increased apoptosis with a significant effect for Q in CO115. Both flavonoids increased p53 expression in both cell lines, an effect particularly remarkable for Q. The significant apoptotic enhancement in CO115 incubated with Q plus 5-FU involved the activation of the apoptotic mitochondrial pathway. Importantly, knockdown of p53 by siRNA in CO115 cells and p53 knockout in HCT116 cells totally abrogated apoptosis induction, demonstrating the dependence of the effect on p53 modulation by Q. This study suggests the potential applicability of these phytochemicals for enhancement 5-FU efficiency in MSI CRC therapy, especially Q in p53 wt tumors.

Tea polyphenol inhibits colorectal cancer with microsatellite instability by regulating the expressions of HES1, JAG1, MT2A and MAFA

To investigate the mechanism of tea polyphenol in inhibiting microsatellite instability (MSI) of colorectal cancer. Using LoVo cells and SW480 cells treated with aqueous solution of tea polyphenol, cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) method, changes in microsatellite sequences were detected by genescan method and changes in gene expression of LoVo cells were detected by illumina expression arrays and quantitative real-time polymerase chain reaction (PCR). The proliferation inhibition rates of LoVo and SW480 cells treated with tea polyphenol increased with the increasing of drug concentration and showed an increasing tendency with time. The proliferation inhibition rate of LoVo cells with tea polyphenol was higher than that of SW480 cells, and there was a significant difference in the proliferation inhibition rates at 24 h, 72 h and one week. The microsatellite sequence of LoVo cells treated with tea polyphenol remained stable. The gene expression arrays and quantitative real-time PCR suggested that tea polyphenol inhibited the gene expressions of MT2A, MAFA, HES1 and JAG1 nearly two-fold over controls. It was also found that tea polyphenol inhibited the BAX and p38 genes with a more than two-fold difference but did not significantly inhibit the nuclear factor-κB pathway. Tea polyphenol significantly inhibited the proliferation of MSI colorectal cancer cells and stably maintained the microsatellite state in MSI colorectal cancer. Tea polyphenol inhibited the gene expressions of HES1, JAG1, MT2A and MAFA, up-regulated the gene expression of BAX and down-regulated that of P38. Further research is required to investigate how these pathways are interrelated.

Abstract A57: Nonsense-mediated decay escaping mutations in microsatellite-unstable colorectal cancer

Abstract Genomic instability drives tumorigenesis by allowing the accumulation of genetic alterations that provide cells with growth advantage. Microsatellite-instability (MSI) and the underlying mutator phenotype caused by a defect in mismatch repair (MMR) functions is the hallmark of Lynch syndrome, and is also observed in a subset of all colorectal cancers (CRC). In cells with a defective MMR system, spontaneous length changes of repetitive microsatellite sequences accumulate all over the genome at highly increased rates. At coding regions instability may lead to frameshift mutations and altered protein products. Genes that mutate this way under MMR deficiency giving selective advantage to cells in tumorigenesis are called MSI target genes. It is generally anticipated that the frameshift mutation-containing transcripts that lead to prematurely terminated proteins undergo nonsense-mediated decay (NMD), followed by a reduction in gene expression levels. However, when a premature stop occurs in the carboxyl-terminal end of the gene it might escape decay mechanisms, which may lead to either dominant-negative or oncogenic effects. Aim of this study was a genome-wide unbiased identification of new MSI CRC target genes that escape NMD. By combining bioinformatic search to expression profiling, we created a list of 330 genes that contained mononucleotide repeats from 6 to 10 base pairs and were likely to be translated despite potential mutations. A novel frameshift predictor software was developed to search all repeat-containing transcripts in the human genome that would escape NMD after one nucleotide deletion. To enhance the odds of identifying oncogenic mutants, the analysis was restricted to genes that were overexpressed in MSI CRC versus normal colonic mucosa. All of these genes were screened initially by sequencing the given repeat in a panel of 30 MSI CRCs.Whenever the mutation frequency exceeded 20% in the tumor set, which was considered evidence for possible selection in MSI tumorigenesis, an additional set of 70 MSI CRCs was sequenced. The great majority of the successfully sequenced genes had no mutations. Altogether four genes were mutated in over 20% of the samples in the extended 100 MSI tumor panel. These candidate driver target genes are being evaluated further by various methods, including sequencing of MSI CRC cell lines and microsatellite-stable (MSS) CRCs, statistical analyses, and functional in vitro experiments. Citation Information: Cancer Res 2009;69(23 Suppl):A57.

High Prevalence of Fatty Acid Synthase Expression in Colorectal Cancers in Middle Eastern Patients and Its Potential Role as a Therapeutic Target

Many human epithelial cancers, particularly those with a poor prognosis, express high levels of fatty acid synthase (FASN), a key metabolic enzyme linked to synthesis of membrane phospholipids in cancer cells. Overexpression of FASN is linked with activation of the phosphatidylinositol-3'-kinase (PI3 K)/AKT pathway. However, the role of FASN in colorectal cancer (CRC) has not been fully elucidated. We investigated the expression of FASN and determined its functional association with the PI3/AKT pathway in CRC. Expression of FASN and its associated targets were studied by immunohistochemistry on 448 CRC tumors in a tissue microarray (TMA) format. Analysis of apoptosis and cell cycle was evaluated in vitro using CRC cell lines by flow cytometry and DNA fragmentation assays. Protein expression was determined by immunohistochemistry and western blotting. In vivo xenograft studies were performed using CRC cell lines and NUDE mice. Correlation of FASN with various clinicopathological parameters on 448 CRC samples was assessed. Activated AKT was found in 294/409 (71.9%) of CRC and was associated with FASN overexpression. FASN expression was observed in 27.1% (109/403) of Middle Eastern CRC. Additionally, FASN expression was significantly more common in tumors characterized by microsatellite instability (MSI) than in those characterized by microsatellite stability (MSS) (P<0.01). Our in vitro data using HCT-15, an MSI CRC cell line, showed a better apoptotic response after inhibition of FASN activity as compared with Colo-320, an MSS CRC cell line. Finally, treatment of HCT-15 cell line xenografts with C-75 resulted in growth inhibition of tumors in NUDE mice via downregulation of FASN and AKT activity. These data identify FASN as a potential biomarker and a novel therapeutic target in distinct molecular subtypes of CRC.

Nonsense-Mediated mRNA Decay Impacts MSI-Driven Carcinogenesis and Anti-Tumor Immunity in Colorectal Cancers

Nonsense-mediated mRNA Decay (NMD) degrades mutant mRNAs containing premature termination codon (PTC-mRNAs). Here we evaluate the consequence of NMD activity in colorectal cancers (CRCs) showing microsatellite instability (MSI) whose progression is associated with the accumulation of PTC-mRNAs encoding immunogenic proteins due to frameshift mutations in coding repeat sequences. Inhibition of UPF1, one of the major NMD factors, was achieved by siRNA in the HCT116 MSI CRC cell line and the resulting changes in gene expression were studied using expression microarrays. The impact of NMD activity was also investigated in primary MSI CRCs by quantifying the expression of several mRNAs relative to their mutational status and to endogenous UPF1 and UPF2 expression. Host immunity developed against MSI cancer cells was appreciated by quantifying the number of CD3ε-positive tumor-infiltrating lymphocytes (TILs). UPF1 silencing led to the up-regulation of 1251 genes in HCT116, among which a proportion of them (i.e. 38%) significantly higher than expected by chance contained a coding microsatellite (P<2×10−16). In MSI primary CRCs, UPF1 was significantly over-expressed compared to normal adjacent mucosa (P<0.002). Our data provided evidence for differential decay of PTC-mRNAs compared to wild-type that was positively correlated to UPF1 endogenous expression level (P = 0.02). A negative effect of UPF1 and UPF2 expression on the host's anti-tumor response was observed (P<0.01). Overall, our results show that NMD deeply influences MSI-driven tumorigenesis at the molecular level and indicate a functional negative impact of this system on anti-tumor immunity whose intensity has been recurrently shown to be an independent factor of favorable outcome in CRCs.

Cytosine Β-D Arabino-Furanoside (Ara-C) and CPT-11 inhibit cell proliferation in human colorectal cancer with microsatellite instability

2061 Background: Recent studies suggest 5-Fluorouracil-based adjuvant chemotherapy does not improve survival in colorectal cancer (CRC) patients with microsatellite instability (MSI) (NEJM, 349 July: 247–257, 2003). We have reported decreased expression of p12DOC-1 is associated with increased proliferation and apoptosis arrest in human CRC with MSI (Oncogene, 22: 6304–10, 2003). The purpose of this study was to measure differential expression of p12DOC-1 using a Cancer Cell Line Profiling (CCLP) Array (Clontech) and to assess the efficacy of CPT-11 and oxaliplatin, in human MSI CRC cell lines. Methods: A CCLP array containing 702 complex cDNA samples from 26 different human cancer cell lines was treated with a range of chemotherapeutic agents. The array was hybridized with a p32-dCTP labeled p12DOC-1 probe. Array results were confirmed using real-time RT-PCR. In addition, the toxicity of CPT-11 and oxaliplatin (neither agent tested on array) against CRC cell lines, RKO (MSI) and SW480 (microsatellite sta...

Cytosine Β-D Arabino-Furanoside (Ara-C) and CPT-11 inhibit cell proliferation in human colorectal cancer with microsatellite instability

2061 Background: Recent studies suggest 5-Fluorouracil-based adjuvant chemotherapy does not improve survival in colorectal cancer (CRC) patients with microsatellite instability (MSI) (NEJM, 349 July: 247–257, 2003). We have reported decreased expression of p12DOC-1 is associated with increased proliferation and apoptosis arrest in human CRC with MSI (Oncogene, 22: 6304–10, 2003). The purpose of this study was to measure differential expression of p12DOC-1 using a Cancer Cell Line Profiling (CCLP) Array (Clontech) and to assess the efficacy of CPT-11 and oxaliplatin, in human MSI CRC cell lines. Methods: A CCLP array containing 702 complex cDNA samples from 26 different human cancer cell lines was treated with a range of chemotherapeutic agents. The array was hybridized with a p32-dCTP labeled p12DOC-1 probe. Array results were confirmed using real-time RT-PCR. In addition, the toxicity of CPT-11 and oxaliplatin (neither agent tested on array) against CRC cell lines, RKO (MSI) and SW480 (microsatellite stable), was measured pre and post transfection with pcDNA3-p12DOC-1. Cell survival was assessed by MTT assay and expressed as IC50 values (50% inhibitory concentration). Results: Ara-C increased p12DOC-1 expression in 2 of 3 MSI CRC cell lines by CCLP array and was confirmed by real-time RT-PCR. Significantly decreased S phase and increased apoptosis was also observed. RT-PCR confirmed an 8-fold increase in p12DOC-1 expression in MSI CRC cell lines after transfection with pcDNA3-p12DOC-1. IC50 values for CPT-11 were significantly higher in all cell lines with increased p12DOC-1 expression (mean IC50= 37 μM), compared to the RKO wild type control (minimal DOC-1 expression) (IC50 =1 μM) (p< 0.05). No differential IC50 values were observed for oxaliplatin. Conclusions: Ara-C induces p12DOC-1 expression, inhibits cell proliferation and increases apoptosis in MSI CRC. In addition, MSI CRC is extremely sensitive to CPT-11. These results suggest that differential gene expression analysis may assist selection of optimal chemotherapeutic agents specifically for the treatment of 5FU resistant MSI CRC. No significant financial relationships to disclose.

Frameshift peptide-derived T-cell epitopes: a source of novel tumor-specific antigens.

Microsatellite instability (MSI) caused by defective DNA mismatch repair (MMR) is a hallmark of hereditary nonpolyposis colorectal cancers (HNPCC) but also occurs in about 15% of sporadic tumors. If instability affects microsatellites in coding regions, translational frameshifts lead to truncated proteins often marked by unique frameshift peptide sequences at their C-terminus. Since MSI tumors show enhanced lymphocytic infiltration and our previous analysis identified numerous coding mono- and dinucleotide repeat-bearing candidate genes as targets of genetic instability, we examined the role of frameshift peptides in triggering cellular immune responses. Using peptide pulsed autologous CD40-activated B cells, we have generated cytotoxic T lymphocytes (CTL) that specifically recognize HLA-A2.1-restricted peptides derived from frameshift sequences. Among 16 frameshift peptides predicted from mutations in 8 different genes, 3 peptides conferred specific lysis of target cells exogenously loaded with cognate peptide. One peptide derived from a (-1) frameshift mutation in the TGFbetaIIR gene gave rise to a CTL bulk culture capable of lysing the MSI colorectal cancer cell line HCT116 carrying this frameshift mutation. Given the huge number of human coding microsatellites and assuming only a fraction being mutated and encoding immunologically relevant peptides in MSI tumors, frameshift protein sequences represent a novel subclass of tumor-specific antigens. It is tempting to speculate that a frameshift peptide-directed vaccination approach not only could offer new treatment modalities for existing MSI tumors but also might benefit asymptomatic at-risk individuals in HNPCC families by a prophylactic vaccination strategy.