Microsatellite Stable Cell Lines Research Articles

PROTAC-mediated conditional degradation of the WRN helicase as a potential strategy for selective killing of cancer cells with microsatellite instability

Multiple studies have demonstrated that cancer cells with microsatellite instability (MSI) are intolerant to loss of the Werner syndrome helicase (WRN), whereas microsatellite-stable (MSS) cancer cells are not. Therefore, WRN represents a promising new synthetic lethal target for developing drugs to treat cancers with MSI. Given the uncertainty of how effective inhibitors of WRN activity will prove in clinical trials, and the likelihood of tumours developing resistance to WRN inhibitors, alternative strategies for impeding WRN function are needed. Proteolysis-targeting chimeras (PROTACs) are heterobifunctional small molecules that target specific proteins for degradation. Here, we engineered the WRN locus so that the gene product is fused to a bromodomain (Bd)-tag, enabling conditional WRN degradation with the AGB-1 PROTAC specific for the Bd-tag. Our data revealed that WRN degradation is highly toxic in MSI but not MSS cell lines. In MSI cells, WRN degradation caused G2/M arrest, chromosome breakage and ATM kinase activation. We also describe a multi-colour cell-based platform for facile testing of selective toxicity in MSI versus MSS cell lines. Together, our data show that a degrader approach is a potentially powerful way of targeting WRN in MSI cancers and paves the way for the development of WRN-specific PROTAC compounds.

Abstract 3095: A high-throughput phenotypic screen to identify small molecule inhibitors of Werner helicase

Abstract Werner helicase (WRN) is a RecQ DNA helicase involved in genome maintenance and DNA damage repair. It has recently been shown to be essential to cancer cells with high microsatellite instability (MSI-H). In MSI-H cancer cells, WRN depletion was shown to induce DNA damage and disrupt mitoses, resulting in a distinct phenotype of enlarged and fragmented nuclei. Here, we present a high-throughput phenotypic screen to identify compounds that selectively induce enlarged nuclei in MSI-H cancer cells. In HCT116, an MSI-H colorectal cancer cell line, approximately 40% of cells exhibited enlarged nuclei when WRN was depleted using siRNA. In contrast, fewer than 10% of control cells displayed this phenotype. In HT29, a microsatellite stable (MSS) cancer cell line, no such difference in nuclear morphology was observed, suggesting that the phenotype is specific to MSI-H cells. We used high content imaging to screen our in-house diversity library of approximately 175,000 compounds in 384-well format. 4,260 compounds induced enlarged nuclei in HCT116 cells, a 2.4% hit rate. After re-screening hits in HCT116 along with two additional MSI-H cancer cell lines, SW48 and SNU-407, and counter-screening in HT29 cells, 144 hits were confirmed to specifically enlarge nuclei in MSI-H but not MSS cells. Using the ADP-Glo Kinase Assay, two of these compounds were confirmed to inhibit WRN ATPase activity, suggesting WRN inhibition as a potential mechanism for their specific effects in MSI-H cells. This screen will also identify compounds which selectively kill MSI-H cells through mechanisms beyond direct inhibition of WRN, including interaction with WRN complex members or other members of DNA damage repair pathways. Validation studies including the assessment of cell viability and DNA damage in a panel of MSI-H and MSS cell lines will be performed. Our study proposes a phenotypic screening strategy for identifying small molecules with the potential to target WRN and uncover additional novel mechanisms for treating MSI-H cancers. Citation Format: Christine A. Moomau, Yipin Lu, Xiaobin Zhang, Xiang Zhai, Lina Gu, Qing Sheng, Fang Li, Yaoyu Chen. A high-throughput phenotypic screen to identify small molecule inhibitors of Werner helicase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3095.

Molecular correlates of sensitivity to PARP inhibition beyond homologous recombination deficiency in pre-clinical models of colorectal cancer point to wild-type TP53 activity.

BackgroundPARP inhibitors are active in various tumour types beyond BRCA-mutant cancers, but their activity and molecular correlates in colorectal cancer (CRC) are not well studied.MethodsMutations and genome-wide mutational patterns associated with homologous recombination deficiency (HRD) were investigated in 255 primary CRCs with whole-exome sequencing and/or DNA copy number data. Efficacy of five PARP inhibitors and their molecular correlates were evaluated in 93 CRC cell lines partly annotated with mutational-, DNA copy number-, and/or gene expression profiles. Post-treatment gene expression profiling and specific protein expression analyses were performed in two pairs of PARP inhibitor sensitive and resistant cell lines.FindingsA subset of microsatellite stable (MSS) CRCs had truncating mutations in homologous recombination-related genes, but these were not associated with genomic signatures of HRD. Eight CRC cell lines (9%) were sensitive to PARP inhibition, but sensitivity was not predicted by HRD-related genomic and transcriptomic signatures. In contrast, drug sensitivity in MSS cell lines was strongly associated with TP53 wild-type status (odds ratio 15.7, p = 0.023) and TP53-related expression signatures. Increased downstream TP53 activity was among the primary response mechanisms, and TP53 inhibition antagonized the effect of PARP inhibitors. Wild-type TP53-mediated suppression of RAD51 was identified as a possible mechanism of action for sensitivity to PARP inhibition.InterpretationPARP inhibitors are active in a subset of CRC cell lines and preserved TP53 function may increase the likelihood of response.

Sensitization of colorectal cancer to irinotecan therapy by PARP inhibitor rucaparib.

Intended to explore synthetic lethality and develop better combinatorial regimens, we screened colorectal cancer (CRC) cells using poly ADP-ribose (PAR) polymerase (PARP) inhibitors and cytotoxic agents. We studied four PARP inhibitors and three DNA-damaging agents, and their combinations using sulforhodamine B assay. Rucaparib demonstrated the greatest synergy with irinotecan, followed by olaparib and PJ34. Rucaparib and irinotecan was further subjected to detailed examination to determine combination index (CI) and underlying mechanism of action. Effectiveness and sequence dependence of this combination were assessed in microsatellite stable (MSS) and unstable (MSI) CRC and HCT116 isogenic cell lines. The degree of cell cycle arrest and apoptosis was determined by FACS. In vivo studies were performed to confirm efficacy of this combination. PAR levels in MSI and PARP expression in MSI and MSS cell lines were diminished upon combinatorial treatment. HCT116 isogenic cells revealed the importance of p21, p53 and PTEN in exerting synergy. In MSI cells, administration of rucaparib prior to irinotecan enhanced cytotoxicity compared to other strategies explored. FACS revealed S-phase arrest and increased late-stage apoptosis in MSS, and G2-M arrest and total and early-stage apoptosis in MSI cells. In in vivo murine xenograft models, a significant reduction in tumor volume and expression of Ki67, pancytokeratin and RPS6KB1, and increase in expression of caspase 3 were observed with the combination. In conclusion, among the various combinations studied, rucaparib plus irinotecan was the most synergistic one. Alterations in cell cycle arrest and apoptosis were dependent on MSI status in CRC cells.

674. Insertional Mutagenesis to Identify Mechanisms of Cetuximab Resistance in Colorectal Cancer

Anti-cancer drugs designed to target specific molecular pathways have shown an excellent therapeutic potential but also very poor long-term durability of tumor responses, mainly due to the outbreak of resistant clones among the residual neoplastic cell population. For that reason, understanding the molecular mechanisms underlying the onset of anti-cancer drug resistance (ACDR) is one of the major goals of clinical research. ACDR has been widely studied by DNA/RNA sequencing of primary human samples and several culprits identified. We have previously developed an approach based on lentiviral vector (LV)-induced insertional mutagenesis that allowed to identify the genes involved in lapatinib and erlotinib resistance on HER2+ human breast cancer cell lines and EGFR+ pancreatic cell line respectively. Here we took advantage of this platform to investigate ACDR genes in colorectal cancer (CRC). Cetuximab, anti-EGFR monoclonal antibody, is used as first line therapy in metastatic CRC, which results in prolonged survival of treated patients. However, nearly all patients relapse due to ACDR. We thus selected CRC cells sensitive to cetuximab deriving either from five microsatellite stable cell lines or from eight Patient Derived Xenografts (PDX), primary human CRC cells implanted subcutaneously into immunodeficient mice (NSG). To induce insertional mutagenesis we generated a luciferase-expressing LV harboring the SFFV enhancer/promoter in the long terminal repeats able to perturb the expression of genes nearby the integration site. As control, we used a non-genotoxic SIN-LV. We set up a collagenase IV-based disaggregation protocol that allows single-cell suspension and a serum-free culture condition to maintain the stemness of in vitro cultured cells. This protocol allowed to efficiently disaggregate and expand CRC cells in vitro as well as reach a LV copy number per cell ranging from 0.25 to 5.6. Luciferase gene expression was stable and allowed live-animal monitoring for up to 30 weeks after transplant. CRC-0069 and -0077 PDXs and NCI-H508 and HDC82 cell lines were transduced ex vivo and kept in vitro and/or transplanted in NSG mice. After in vitro or in vivo expansion of the transduced CRCs cetuximab treatment was applied. After an initial shrinking of the tumor mass in mice we observed ACDR in 3 out of 10 mice transplanted with NCI-H508 cells transduced with SFFV-LV and in none of the controls. Genomic DNA from resistant cells is being used for insertion site (IS) analysis to identify common IS, ACDR gene candidates. IS obtained from SIN-LV groups will be used to filter LV integration biases, whereas IS from SFFV-LV transduced cells but not treated with cetuximab will be used to filter mutations that provide a proliferative advantage unrelated to cetuximab treatment. We will validate the most promising candidates by LV-mediated overexpression and knockdown techniques. This approach could pave the way to perform insertional mutagenesis-based forward genetics studies on primary human samples.

Instability of a dinucleotide repeat in the 3′-untranslated region (UTR) of the microsomal prostaglandin E synthase-1 (mPGES-1) gene in microsatellite instability-high (MSI-H) colorectal carcinoma

DNA mismatch-repair gene mutations, with consequent loss of functional protein expression, result in microsatellite instability (MSI). Microsatellite sequences are found in coding regions and in regulatory regions of genes (i.e., 5′-UTRs and 3′-UTRs). In addition to being a surrogate marker of defective mismatch repair, deletion or insertion microsatellite sequences can dysregulate gene expression in MSI-H (microsatellite instability-high) tumors. The microsomal prostaglandin E synthase-1 (mPGES-1) gene product, mPGES-1, participates in prostaglandin E2 (PGE2) production. Moreover, mPGES-1 is often overexpressed in human colorectal tumors, and is thought to contribute to progression of these tumors. Here we identified a dinucleotide repeat, (GT)24, in the mPGES-1 gene 3′ untranslated region (3′-UTR), and analyzed its mutation frequencies in MSI-H and microsatellite stable (MSS) tumors. The (GT)24 repeat exhibited instability in all MSI-H tumors examined (14), but not in any of the MSS tumors (13). In most cases, (GT)24 repeat instability resulted in insertion of additional GT units. We also determined mPGES-1 mRNA levels in MSI-H and MSS colorectal cancer cell lines. Three of four previously designated “MSI-H” cell lines showed higher mPGES-1 mRNA levels compared to MSS cell lines; correlations between elevated mPGES-1 mRNA levels and microsatellite (GT)24 repeat characteristics are present for all six cell lines. Our results demonstrate that mPGES-1 is a target gene of defective mismatch repair in human colorectal cancer, with functional consequence.

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.

Expression of the MAP kinase phosphatase DUSP4 is associated with microsatellite instability in colorectal cancer (CRC) and causes increased cell proliferation

DUSP4 (MKP-2), a member of the mitogen-activated protein kinase phosphatase (MKP) family and potential tumor suppressor, negatively regulates the MAPKs (mitogen-activated protein kinases) ERK, p38 and JNK. MAPKs play a crucial role in cancer development and progression. Previously, using microarray analyses we found a conspicuously frequent overexpression of DUSP4 in colorectal cancer (CRC) with high frequent microsatellite instability (MSI-H) compared to microsatellite stable (MSS) CRC. Here we studied DUSP4 expression on mRNA level in 38 CRC (19 MSI-H and 19 MSS) compared to matched normal tissue as well as in CRC cell lines by RT-qPCR. DUSP4 was overexpressed in all 19 MSI-H tumors and in 14 MSS tumors. Median expression levels in MSI-H tumors were significantly higher than in MSS-tumors (p < 0.001). Consistently, MSI-H CRC cell lines showed 6.8-fold higher DUSP4 mRNA levels than MSS cell lines. DUSP4 expression was not regulated by promoter methylation since no methylation was found by quantitative methylation analysis of DUSP4 promoter in CRC cell lines neither in tumor samples. Furthermore, no DUSP4 mutation was found on genomic DNA level in four CRC cell lines. DUSP4 overexpression in CRC cell lines through DUSP4 transfection caused upregulated expression of MAPK targets CDC25A, CCND1, EGR1, FOS, MYC and CDKN1A in HCT116 as well as downregulation of mismatch repair gene MSH2 in SW480. Furthermore, DUSP4 overexpression led to increased proliferation in CRC cell lines. Our findings suggest that DUSP4 acts as an important regulator of cell growth within the MAPK pathway and causes enhanced cell growth in MSI-H CRC.

Differential expression of CD133 based on microsatellite instability status in human colorectal cancer

The association between the types of genomic instability and cancer stem cell (CSC) has not been elucidated. We aimed to investigate the expressions of CSC markers with respect to microsatellite instability (MSI) status in human colorectal cancer (CRC). Immunostainings for CD133, CD44, and CD166, and K-ras mutation analysis were performed on 50 MSI-high (MSI-H), and 50 microsatellite stable (MSS) CRC tissues. In 11 MSS and MSI-H CRC cell lines, CD133 expression and DNA methylation statuses of the CD133 promoter were determined. The proportion of CD133 positive cells and the ability of colosphere formation were compared between HCT116 cells and HCT116 + Chr3 cells (hMLH1-restored HCT116 cells). Immunohistochemistry for CSC markers revealed that high CD133 expression was more frequent in MSS cancers than in MSI-H (P < 0.001, 74.0% vs. 28.0%, respectively), and related with short disease-free survival. Neither CD44 nor CD166 expression differed significantly with respect to MSI status. K-ras mutation showed no association with expressions of CD133, CD44, or CD166. CD133 expression was relatively high in the MSS cell lines compared to those in MSI-H, and showed a reverse correlation with DNA methylation of the CD133 promoter. hMLH1-restored HCT116 cells increased proportions of CD133 positive cells and colosphere forming ability, compared to those in HCT116 cells. In conclusion, high levels of CD133 expression were observed more frequently in MSS CRC than in MSI-H, suggesting that differential expression of colon CSC markers may be linked to tumor characteristics dependent on MSI status.

Gene Expression Variations in Microsatellite Stable and Unstable Colon Cancer Cells

Microsatellite instability (MSI) is a marker of chemoresistance, but it is associated with improved survival compared with microsatellite-stable (MSS) colon cancers. We hypothesized that MSI tumors overexpress chemoresistance-associated genes and underexpress DNA damage/repair genes. We used ultra high-throughput sequencing (UHTS) to assess the expression of representative genes in MSI and MSS colon cancer cell lines. Solexa UHTS was used to examine gene expression in HCT116 (MSI) and HT29 (MSS) cells, and normal colonic mucosa (NCM). We compared expression of 40 genes involved in chemoresistance, DNA repair, DNA damage, and drug metabolism pathways. We observed gene expression differences between MSI and MSS cell lines in 8 out of 40 genes involved in mismatch repair (MMR), DNA repair, drug metabolism, and chemoresistance. MMR gene expression was lower in MSI cells, which is consistent with the MSI phenotype, whereas DNA repair genes were highly expressed in these cells. Genes associated with chemoresistance and drug metabolism also had increased expression in MSI cells. No difference in expression of DNA damage genes was observed between MSI and MSS cell lines. Using UHTS gene expression analysis, we identified differential expression of genes between MSI and MSS cell lines which may account for resistance to chemotherapy in MSI tumors. UHTS expression analysis has the potential to identify genome-wide predictors of response or resistance to chemotherapy.

Preclinical testing of the PARP inhibitor ABT-888 in microsatellite instable colorectal cancer

11028 Background: Microsatellite instability (MSI) represents approximately 15% of colorectal cancer (CRC) cases. MSI due to hypermethylation or mutation in DNA mismatch repair genes leads to genetic instability and a mutator phenotype. Genetic instability is particularly high at repetitive sequences such as those located in MRE11, RAD50, CtIP and MBC. Each of these genes are implicated in the double strand break (DSB) repair pathway. PARP inhibitors induce single strand breaks that remain unrepaired and then will be converted to DSB during DNA replication. Our objective was to assess the preclinical activity of a novel PARP inhibitor ABT-888 in MSI cell lines deficient in the DSB repairing pathway and compare it to Microsatellite Stable (MSS) lines. Methods: We used the systems biology tool “Connectivity Map” to synthesize data from 5 different published studies of expression profiling of MSI CRC phenotype and to identify target compounds. We assessed the mutational status of MRE11, RAD50, CtIP and MBC in a panel of 10 CRC cell lines displaying either MSI or MSS, and measured the expression of MRE11 by quantitative RT-PCR. We tested the cytotoxic activity of single-agent ABT-888 for 6 days in MSS and MSI cell lines, stratified by mutational status. Flow cytometry was performed after 24 hours. Results: Systems biology studies identified PARP inhibitors as a candidate compound relevant for MSI CRC. Mutational status of MRE11 was perfectly correlated with MSI status. ABT-888 shows a preferential activity on those MSI cell lines harboring mutations in both MRE11 and RAD50 genes compared to MSS cell lines (wild-type for both genes). A significant correlation exists between MRE11 expression levels and cytotoxicity to ABT-888 at 10 μM (R2=0.915, P&lt;0.001). Flow cytometry analyses show a G1 arrest following to the treatment with ABT-888 that is higher in MSI cell lines with mutations in MRE11 and RAD50 compared to MSS cell lines. Conclusions: This is the first report of the preclinical activity of a PARP inhibitor in CRC models. MSI colorectal tumors deficient in DSB repair show a higher sensitivity to PARP inhibition. Further clinical investigation of ABT-888 as a single agent or in combination with other chemotherapy drugs inducing DSB is warranted in MSI CRC with mutations in MRE11 and RAD50. No significant financial relationships to disclose.

The Bone Morphogenetic Protein Pathway Is Inactivated in the Majority of Sporadic Colorectal Cancers

The finding of bone morphogenetic protein (BMP) receptor 1a mutations in juvenile polyposis suggests that BMPs are important in colorectal cancer (CRC). We investigated the BMP pathway in sporadic CRC. We investigated BMP receptor (BMPR) expression using immunoblotting and sequenced BMPR2 in CRC cell lines. We assessed the expression of BMPRs, SMAD4, and pSMAD1/5/8 in 72 sporadic CRCs using a tissue microarray and immunohistochemistry. We assessed the effect of reintroduction of wild-type BMPR2 on BMP pathway activity and the effect of wild-type or mutated BMPR2 3' untranslated region (UTR) sequences on protein expression by attachment to pCMV-Luc. BMPR2 and SMAD4 protein expression is abrogated in microsatellite unstable (MSI) and microsatellite stable (MSS) cell lines, respectively. BMPR2 3'UTR is mutated in all MSI and in none of the MSS cell lines. Mutant BMPR2 3'UTR sequences reduced luciferase expression 10-fold compared with wild-type BMPR2 3'UTR. BMPR2 expression is impaired more frequently in MSI CRCs than MSS (85% vs 29%; P < .0001) and shows a mutually exclusive pattern of impaired expression compared with SMAD4. Nine of 11 MSI cancers with impaired expression of BMPR2 have microsatellite mutations. The BMP pathway is inactivated, as judged by nuclear pSMAD1/5/8 expression, in 70% of CRCs, and this correlates with BMPR and SMAD4 loss. Our data suggest that the BMP pathway is inactivated in the majority of sporadic CRCs. In MSI CRC this is associated predominantly with impaired BMPR2 expression and in MSS CRC with impaired SMAD4 expression.

Phosphoprotein Keratin 23 accumulates in MSS but not MSI colon cancers in vivo and impacts viability and proliferation in vitro

Transcript profiling of 27 normal colon mucosas and 258 adenocarcinomas showed Keratin23 to be increased in 78% microsatellite-stable tumors, while microsatellite-instable tumors showed low transcript levels, comparable to normal mucosas. Immunohistochemical analyses demonstrated that 88% of microsatellite-instable tumors were negative for Keratin23 protein, while 70% of MSS tumors and metastases derived from MSS-tumors showed high Keratin23 levels. Immunofluorescence analysis localized Keratin23 in the Golgi-apparatus. Golgi accumulation was unique for gastrointestinal adenocarcinomas. Immunoprecipitation and 2D-blot analysis revealed Keratin23 to be a 46.8 kDa phosphoprotein. Keratin23 impaired the proliferation of human colon cancer cells significantly, leading to cell death in microsatellite-instable but not microsatellite-stable cell lines, while COS7 cells experienced multiple nuclei and apoptosis. Keratin23 expression correlated significantly with transcription factor CEBPB. In conclusion, Keratin23 expression is a novel and important difference between microsatellite-stable and microsatellite-instable colon cancers.

Differential expression of DHHC9 in microsatellite stable and instable human colorectal cancer subgroups

Microarray analysis on pooled samples has previously identified ZDHHC9 (DHHC9) to be upregulated in colon adenocarcinoma compared to normal colon mucosa. Analyses of 168 samples from proximal and distal adenocarcinomas using U133plus2.0 microarrays validated these findings, showing a significant two-fold (log 2) upregulation of DHHC9 transcript (P<10−6). The upregulation was more striking in microsatellite stable (MSS), than in microsatellite instable (MSI), tumours. Genes known to interact with DHHC9 as H-Ras or N-Ras did not show expression differences between MSS and MSI. Immunohistochemical analysis was performed on 60 colon adenocarcinomas, previously analysed on microarrays, as well as on tissue microarrays with 40 stage I–IV tumours and 46 tumours from different organ sites. DHHC9 protein was strongly expressed in MSS compared to MSI tumours, readily detectable in premalignant lesions, compared to the rare expression seen in normal mucosa. DHHC9 was specific for tumours of the gastrointestinal tract and localised to the Golgi apparatus, in vitro and in vivo. Overexpression of DHHC9 decreased the proliferation of SW480 and CaCo2 MSS cell lines significantly. In conclusion, DHHC9 is a gastrointestinal-related protein highly expressed in MSS colon tumours. The palmitoyl transferase activity, modifying N-Ras and H-Ras, suggests DHHC9 as a target for anticancer drug design.

P59: Microsatellite unstable (MSI) colorectal cancer (CRC) is characterized by significantly increased epidermal growth factor receptor (EGFR) expression

Introduction: EGFR over expression has been reported in multiple solid tumor malignancies including colorectal cancer and the EGFR targeted molecular therapy Cetuximab, has demonstrated significant but modest effectiveness against human CRC. Importantly, literature suggests microsatellite unstable (MSI) CRC is characterized by poor response to standard adjuvant chemotherapy compared to microsatellite stable (MSS) CRC. The purpose of our present study was to expand our analysis of differential gene expression in human CRC (Oncogene 2005 24:3657-3668) and test the hypothesis that MSI CRC is characterized by significantly increased expression of EGFR over that observed in microsatellite stable (MSS) CRC. Methods: Twenty eight human CRC cell lines including 11MSI cell lines were employed to measure EGFR protein and mRNAexpression using properly controlled Western blots and real time PCR respectively. Independently read densitometry analysis of those same Western blots and quantitative analysis of the RT PCR data was performed. Results: EGFR expression was detected by Western blot and RT PCR in each of the 28 CRC cell lines studied including the 11 MSI lines. Comparative analysis of the MSI and MSS CRC cell line results demonstrated significantly increased EGFR expression among the MSI lines over that observed in the MSS lines as measured by Western blot and RT PCR. Densitometry analysis of these same Western blots and quantitative analysis of the RT PCR data confirmed statistically significant increased EGFR expression levels among MSI vs. MSS cell lines with p values of 0.001 and 0.009 respectively. Conclusions: In summary, these novel results support our proposed hypothesis indicating MSI CRC is characterized by significantly increased EGFR expression over that observed in MSS CRC. These results suggest EGFR targeted molecular therapies may provide important alternative therapeutic strategies for MSI CRC. Based on these findings we conclude further study of EGFR over expression in MSI CRC is warranted and an expanded analysis of the mechanism(s) of increased EGFR expression in human MSI CRC is underway in our laboratory.

Chemotherapeutic implications in microsatellite unstable colorectal cancer.

Chemotherapy for colorectal cancer is currently offered to patients based on the stage of their cancer, and there is evidence to show an overall survival benefit with 5-fluorouracil-based (5-FU) therapy for patients with lymph node metastasis who receive it. The pathogenesis of colorectal cancer involves genomic instability, with about 15% of tumors demonstrating a form of genomic instability called high-frequency microsatellite instability (MSI-H) and due to loss of DNA mismatch repair function, and the remainder of colorectal tumors lacking MSI-H with retained DNA mismatch repair function and called microsatellite stable (MSS), with a large proportion of these tumors demonstrating another form of genomic instability called chromosomal instability. There is now evidence to show that the form of genomic instability that is present in a patient's colorectal cancer may predict a survival benefit from 5-FU. In particular, patients whose colorectal tumors have MSI-H do not gain a survival benefit with 5-FU as compared to patients with MSS tumors. In vitro evidence supports these findings, as MSI-H colon cancer cell lines are more resistant to 5-FU compared to MSS cell lines. More specifically, components of the DNA mismatch repair system have been shown to recognize and bind to 5-FU that becomes incorporated into DNA and which could be a trigger to induce cell death. The binding and subsequent cell death events would be absent in colorectal tumors with MSI-H, which have lost intact DNA mismatch repair function. These findings suggest that: (a) tumor cytotoxicity of 5-FU is mediated by DNA mechanisms in addition to well-known RNA mechanisms, and (b) patients whose tumors demonstrate MSI-H may not benefit from 5-FU therapy. Future studies should include a better understanding of the cellular mechanisms of the DNA recognition of 5-FU, multi-centered prospective trials investigating the survival benefit of 5-FU based on genomic instability, and the investigation of alternative chemotherapeutic regimens for patients with MSI-H tumors to improve survival.