Joongho Shin

PIK3CA Mutation/PTEN Expression Status Predicts Response of Colon Cancer Cells to the Epidermal Growth Factor Receptor Inhibitor Cetuximab

Cetuximab is a monoclonal antibody that targets the human epidermal growth factor receptor (EGFR). Although approved for use in EGFR-overexpressing advanced colorectal cancer, recent studies have shown a lack of association between EGFR overexpression and cetuximab response, requiring the identification of novel biomarkers predictive of response to this agent. To do so, 22 colon cancer cell lines were screened for cetuximab response in vitro and sensitive and resistant lines were identified. In sensitive cell lines, cetuximab induced a G(0)-G(1) arrest without inducing apoptosis. Notably, cetuximab-sensitive but not cetuximab-resistant cell lines were preferentially responsive to EGF-stimulated growth. Whereas neither EGFR protein/mRNA expression nor gene copy number correlated with cetuximab response, examination of the mutation status of signaling components downstream of EGFR showed that cell lines with activating PIK3CA mutations or loss of PTEN expression (PTEN null) were more resistant to cetuximab than PIK3CA wild type (WT)/PTEN-expressing cell lines (14 +/- 5.0% versus 38.5 +/- 6.4% growth inhibition, mean +/- SE; P = 0.008). Consistently, PIK3CA mutant isogenic HCT116 cells showed increased resistance to cetuximab compared with PIK3CA WT controls. Furthermore, cell lines that were PIK3CA mutant/PTEN null and Ras/BRAF mutant were highly resistant to cetuximab compared with those without dual mutations/PTEN loss (10.8 +/- 4.3% versus 38.8 +/- 5.9% growth inhibition, respectively; P = 0.002), indicating that constitutive and simultaneous activation of the Ras and PIK3CA pathways confers maximal resistance to this agent. A priori screening of colon tumors for PTEN expression status and PIK3CA and Ras/BRAF mutation status could help stratify patients likely to benefit from this therapy.

An A13 Repeat within the 3′-Untranslated Region of Epidermal Growth Factor Receptor (EGFR) Is Frequently Mutated in Microsatellite Instability Colon Cancers and Is Associated with Increased EGFR Expression

Colorectal cancers (CRC) with microsatellite instability (MSI) have clinical, pathologic, genetic, and epigenetic features distinct from microsatellite-stable CRC. Examination of epidermal growth factor receptor (EGFR) mRNA and protein expression levels in a panel of colon cancer cell lines identified strong expression of EGFR in multiple cell lines with MSI. Although no relationship between EGFR overexpression and the length of a CA dinucleotide repeat in intron 1 was observed, a variant A13/A14 repeat sequence within the 3-untranslated region (3-UTR) of the EGFR gene was identified, which was mutated by either mononucleotide or dinucleotide adenosine deletions in 64% of MSI cell lines and 69% of MSI colon tumors. Using a Tet-Off system, we show that this mutation increases EGFR mRNA stability in colon cancer cells, providing a mechanistic basis for EGFR overexpression in MSI colon cancer cell lines. To determine whether this mutation is a driver or a bystander event in MSI colon cancer, we examined the effect of pharmacologic and molecular inhibition of EGFR in EGFR 3-UTR mutant MSI cell lines. Cell lines with an EGFR 3-UTR mutation and that were wild-type (WT) for downstream signaling mediators in the Ras/BRAF and PIK3CA/PTEN pathways were sensitive to EGFR inhibition, whereas those harboring mutations in these signaling mediators were not. Furthermore, in cell lines WT for downstream signaling mediators, those with EGFR 3-UTR mutations were more sensitive to EGFR inhibition than EGFR 3-UTR WT cells, suggesting that this mutation provides a growth advantage to this subset of MSI colon tumors.

The intestinal epithelial cell differentiation marker intestinal alkaline phosphatase (ALPi) is selectively induced by histone deacetylase inhibitors (HDACi) in colon cancer cells in a Kruppel-like factor 5 (KLF5)-dependent manner.

Background: Differentiation induction represents a potential cancer treatment strategy. Results: Colon cancer cell lines respond differentially to HDACi-mediated induction of the differentiation marker ALPi. HDACi induction of ALPi is KLF5-dependent. Conclusion: HDACi induce ALPi in a subset of colon cancer cell lines in a KLF5-dependent manner. Significance: Colon cancer cell lines are differentially responsive to HDACi-induced differentiation. The histone deacetylase inhibitor (HDACi) sodium butyrate promotes differentiation of colon cancer cells as evidenced by induced expression and enzyme activity of the differentiation marker intestinal alkaline phosphatase (ALPi). Screening of a panel of 33 colon cancer cell lines identified cell lines sensitive (42%) and resistant (58%) to butyrate induction of ALP activity. This differential sensitivity was similarly evident following treatment with the structurally distinct HDACi, MS-275. Resistant cell lines were significantly enriched for those harboring the CpG island methylator phenotype (p = 0.036, Chi square test), and resistant cell lines harbored methylation of the ALPi promoter, particularly of a CpG site within a critical KLF/Sp regulatory element required for butyrate induction of ALPi promoter activity. However, butyrate induction of an exogenous ALPi promoter-reporter paralleled up-regulation of endogenous ALPi expression across the cell lines, suggesting the presence or absence of a key transcriptional regulator is the major determinant of ALPi induction. Through microarray profiling of sensitive and resistant cell lines, we identified KLF5 to be both basally more highly expressed as well as preferentially induced by butyrate in sensitive cell lines. KLF5 overexpression induced ALPi promoter-reporter activity in resistant cell lines, KLF5 knockdown attenuated butyrate induction of ALPi expression in sensitive lines, and butyrate selectively enhanced KLF5 binding to the ALPi promoter in sensitive cells. These findings demonstrate that butyrate induction of the cell differentiation marker ALPi is mediated through KLF5 and identifies subsets of colon cancer cell lines responsive and refractory to this effect.

Intestinal epithelial-specific PTEN inactivation results in tumor formation

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of phosphatidylinositol 3-kinase (PI3K) signaling that is frequently inactivated in colorectal cancer through mutation, loss of heterozygosity, or epigenetic mechanisms. The aim of this study was to determine the effect of intestinal-specific PTEN inactivation on intestinal epithelial homeostasis and tumorigenesis. PTEN was deleted specifically in the intestinal epithelium, by crossing PTEN(Lox/Lox) mice with villin(Cre) mice. PTEN was robustly expressed in the intestinal epithelium and maximally in the differentiated cell compartment. Targeted inactivation of PTEN in the intestinal epithelium of PTEN(Lox/Lox)/villin(Cre) mice was confirmed by genotyping, immunohistochemistry, and qPCR. While intestinal-specific PTEN deletion did not have a major effect on cell fate determination or proliferation in the small intestine, it did increase phosphorylated (p) protein kinase B (AKT) expression in the intestinal epithelium, and 19% of animals developed small intestinal adenomas and adenocarcinomas at 12 mo of age. These tumors demonstrated pAKT and nuclear β-catenin staining, indicating simultaneous activation of the PI3K/AKT and Wnt signaling pathways. These findings demonstrate that, while PTEN inactivation alone has a minimal effect on intestinal homeostasis, it can facilitate tumor promotion upon deregulation of β-catenin/TCF signaling, further establishing PTEN as a bona fide tumor suppressor gene in intestinal cancer.

Correction: The intestinal epithelial cell differentiation marker intestinal alkaline phosphatase (ALPi) is selectively induced by histone deacetylase inhibitors (HDACi) in colon cancer cells in a Kruppel-like factor 5 (KLF5)-dependent manner (Journal of Biological Chemistry)

Joongho Shin, Azadeh Carr, Georgia A. Corner, Lars Tögel, Mercedes Dávaos-Salas, Hoanh Tran, Anderly C. Chueh, Sheren Al-Obaidi, Fiona Chionh, Naseem Ahmed, Daniel D. Buchanan, Joanne P. Young, Madhu S. Malo , Richard A. Hodin , Diego Arango**, Oliver M. Sieber, Leonard H. Augenlicht, Amardeep S. Dhillon, Thomas K. Weber, and John M. Mariadason From the Department of Oncology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York 10461, the Ludwig Institute for Cancer Research, Austin Health, Melbourne 3084, Australia, the Queensland Institute of Medical Research, 300 Herston Road, Herston, Queensland 4006, Australia, the Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02115, the **Group of Molecular Oncology, Centro en Investigación en Bioquı́mica y Biologı́a Molecular-Nanomedicine, Vall d’Hebron University Hospital, Research Institute, Universitat Autònoma de Barcelona, Passeig Vall d’Hebron, 119-129, 08035 Barcelona, Spain and El Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina, Spain, the Walter and Eliza Hall Institute, Melbourne 3052, Australia, Peter MacCallum Cancer Center, Melbourne 3002, Australia, and the Veterans Affairs New York Harbor Health Care System, Brooklyn, New York 11209

Abstract 2991: Coordinate methylation of the alkaline phosphatase gene family in colon cancer. Implications for understanding the manifestation of the CpG island methylator phenotype (CIMP). .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnThe HDAC-inhibitor sodium-butyrate, promotes differentiation of colon cancer cells as evidenced by induction of alkaline phosphatase (ALP) enzyme activity. Screening of a panel of 34 colon cancer cell lines identified cell lines responsive (44%) and refractory (56%) to butyrate-induced differentiation. Refractory cell lines were significantly enriched for those harbouring the CpG island methylator phenotype (CIMP) (P=0.016). The ALP gene family comprises 4 members - ALPi, ALPL, ALPP and ALPPL2 - each of which was induced by butyrate in responsive cell lines, demonstrating co-ordinate regulation of this gene family in response to HDACi treatment. Deletion analyses of the ALPi promoter identified a KLF/Sp1 cis element essential for butyrate-induction of ALPi promoter activity. Remarkably, we observed that a CpG dinucleotide within this site was preferentially methylated in refractory cell lines. Furthermore, the majority of cell lines with methylation of this site in the ALPi promoter also harboured methylation of similar sites within the ALPL and ALPP promoters, indicating coordinate methylation of this gene family in refractory cell lines. Frequent methylation and reduced expression of ALPi and ALPL were also observed in primary human colon cancers. Targeted inactivation of the DNA methyltransferases Dnmt1 and Dnmt3b (HCT116-DKO) and co-treatment with decitabine restored butyrate-inducibility of ALP in the methylated HCT116 cell line. However, butyrate-induction of an exogenous ALPi promoter-reporter paralleled induction of endogenous ALPi mRNA across the cell lines, suggesting ALPi promoter methylation may not be the primary determinant of ALP inducibility. We therefore explored whether the differential induction of the ALP gene family in colon cancer cells was due to selective induction of a common transcriptional regulator. To address this, responsive and refractory cell lines were treated with butyrate for 72h and transcription factors selectively induced in responsive cell lines identified by microarray. By focussing on factors which bind KLF/Sp1 regulatory elements, we identified KLF5 as selectively induced in responsive cell lines. KLF5 was also preferentially induced by butyrate in HCT116 DKO cells. Importantly, knockdown of KLF5 markedly attenuated butyrate-induction of ALP mRNA expression and enzyme activity. These findings demonstrate that the ALP gene family is co-ordinately inducible by butyrate in select colon cancer cell lines, in a KLF5-dependent manner. Cells lines refractory to butyrate-induction of the ALP gene family were enriched for CIMP high lines, and displayed coordinate promoter methylation. We propose that the coordinate methylation of this gene family in CIMP high colon cancers is likely linked to their coordinate regulation.nnCitation Format: John Mariadason, Azadeh Carr, Georgia A. Corner, Sheren Al-Obaidi, Anderly Chueh, Fiona Chionh, Lars Togel, Naseem Ahmed, Diego Arango, Daniel Buchanan, Joanne Young, Leonard H. Augenlicht, Madhu S. Malo, Richard Hodin, Oliver M. Sieber, Thomas Weber, Joongho Shin. Coordinate methylation of the alkaline phosphatase gene family in colon cancer. Implications for understanding the manifestation of the CpG island methylator phenotype (CIMP). . [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 2991. doi:10.1158/1538-7445.AM2013-2991

Abstract 4921: Selective promoter methylation of the cell differentiation marker, intestinal alkaline phosphatase (iALP), in microsatellite unstable colon cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnTumorigenesis is characterized by excessive cell growth, resistance to apoptosis and aberrant cellular differentiation. In particular, MSI colorectal cancer is frequently characterized by poorly differentiated histology. Histone Deacetylase inhibitors (HDACi) are a novel class of cancer therapeutics that induce growth arrest, apoptosis and differentiation of a variety of tumour types, including colon cancer cells. Differentiation inducing agents have been used effectively to treat hematopoietic malignancies, prompting us to examine the effects of HDACi treatment on differentiation of colon cancer cells, and to compare the effects induced in MSS and MSI colon cancers. Remarkably, while HDACi treatment robustly and consistently induced differentiation induction as measured by alkaline phosphatise activity and iALP gene expression in multiple MSS colon cancer cell lines, approximately 70% of MSI colon cancer cell lines were highly resistant to HDACi-induced ALP induction. Examination of the coding sequence of the iALP gene failed to identify any significant repeat elements suggesting mutation is unlikely to be responsible for lack of iALP induction. Furthermore, transient transfection of an iALP promoter reporter construct demonstrated robust induction in response to butyrate treatment in both MSS and MSI lines, suggesting loss of a key regulatory factor in MSI cell lines is not likely to be responsible. Conversely, examination of iALP promoter methylation by bisulphite conversion and direct sequencing demonstrated multiple CpG sites, including a Sp1/Sp3 binding site, to be methylated in 4 of 5 MSI lines examined, while no methylation was observed in MSS lines. These findings were confirmed using the sequenom assay. ALP promoter methylation was independent of CIMP status. The importance of Sp1/Sp3 in butyrate-induced iALP induction was demonstrated by the ability of the Sp1/Sp3 inhibitor, mithramycin, to attenuate butyrate-induced ALP activity. Finally, consistent with methylation being a key determinant of iALP expression in MSI lines, azacytidine treatment was able to re-induce iALP expression, and butyrate-induction of ALP expression was markedly enhanced in isogenic HCT116 cells lacking the DNA methyltransferases Dnmt1 and Dnmt3b (DKO), compared to parental cells. These findings demonstrate selective promoter methylation of iALP in MSI colon cancer cell lines.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4921.