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Supplementary Figure from Ovarian Cancers with Low CIP2A Tumor Expression Constitute an APR-246–Sensitive Disease Subtype

Identification of ovarian cancer patient subpopulations with increased sensitivity to targeted therapies could offer significant clinical benefit. We report that 22% of the high-grade ovarian cancer tumors at diagnosis express CIP2A oncoprotein at low levels. Furthermore, regardless of their significantly lower likelihood of disease relapse after standard chemotherapy, a portion of relapsed tumors retain their CIP2A-deficient phenotype. Through a screen for therapeutics that would preferentially kill CIP2A-deficient ovarian cancer cells, we identified reactive oxygen species inducer APR-246, tested previously in ovarian cancer clinical trials. Consistent with CIP2A-deficient ovarian cancer subtype in humans, CIP2A is dispensable for development of MISIIR-Tag–driven mouse ovarian cancer tumors. Nevertheless, CIP2A-null ovarian cancer tumor cells from MISIIR-Tag mice displayed APR-246 hypersensitivity both in vitro and in vivo. Mechanistically, the lack of CIP2A expression hypersensitizes the ovarian cancer cells to APR-246 by inhibition of NF-κB activity. Accordingly, combination of APR-246 and NF-κB inhibitor compounds strongly synergized in killing of CIP2A-positive ovarian cancer cells. Collectively, the results warrant consideration of clinical testing of APR-246 for CIP2A-deficient ovarian cancer tumor subtype patients. Results also reveal CIP2A as a candidate APR-246 combination therapy target for ovarian cancer.

Supplementary Table from Ovarian Cancers with Low CIP2A Tumor Expression Constitute an APR-246–Sensitive Disease Subtype

Supplementary Data from Ovarian Cancers with Low CIP2A Tumor Expression Constitute an APR-246–Sensitive Disease Subtype

PDF file - 66K, Supplementary Table S1. Co-relation between Chk1 and c-MYC nuclear staining in gastric cancer.

PDF file - 85K, Figure S2. Chk1 and Claspin (CLSPN) positively regulate CIP2A protein expression in human cancer cells.

Supplementary Figures 1-6, Tables 1-3 from Oncolytic Adenovirus Coding for Granulocyte Macrophage Colony-Stimulating Factor Induces Antitumoral Immunity in Cancer Patients

Checkpoint kinase Chk1 is constitutively active in many cancer cell types and new generation Chk1 inhibitors show marked antitumor activity as single agents. Here we present a hitherto unrecognized mechanism that contributes to the response of cancer cells to Chk1-targeted therapy. Inhibiting chronic Chk1 activity in cancer cells induced the tumor suppressor activity of protein phosphatase protein phosphatase 2A (PP2A), which by dephosphorylating MYC serine 62, inhibited MYC activity and impaired cancer cell survival. Mechanistic investigations revealed that Chk1 inhibition activated PP2A by decreasing the transcription of cancerous inhibitor of PP2A (CIP2A), a chief inhibitor of PP2A activity. Inhibition of cancer cell clonogenicity by Chk1 inhibition could be rescued in vitro either by exogenous expression of CIP2A or by blocking the CIP2A-regulated PP2A complex. Chk1-mediated CIP2A regulation was extended in tumor models dependent on either Chk1 or CIP2A. The clinical relevance of CIP2A as a Chk1 effector protein was validated in several human cancer types, including neuroblastoma, where CIP2A was identified as an NMYC-independent prognostic factor. Because the Chk1–CIP2A–PP2A pathway is driven by DNA-PK activity, functioning regardless of p53 or ATM/ATR status, our results offer explanative power for understanding how Chk1 inhibitors mediate single-agent anticancer efficacy. Furthermore, they define CIP2A-PP2A status in cancer cells as a pharmacodynamic marker for their response to Chk1-targeted therapy. Cancer Res; 73(22); 6757–69. ©2013 AACR.

Granulocyte macrophage colony-stimulating factor (GMCSF) can mediate antitumor effects by recruiting natural killer cells and by induction of tumor-specific cytotoxic T-cells through antigen-presenting cells. Oncolytic tumor cell–killing can produce a potent costimulatory danger signal and release of tumor epitopes for antigen-presenting cell sampling. Therefore, an oncolytic adenovirus coding for GMCSF was engineered and shown to induce tumor-specific immunity in an immunocompetent syngeneic hamster model. Subsequently, 20 patients with advanced solid tumors refractory to standard therapies were treated with Ad5-D24-GMCSF. Of the 16 radiologically evaluable patients, 2 had complete responses, 1 had a minor response, and 5 had disease stabilization. Responses were frequently seen in injected and noninjected tumors. Treatment was well tolerated and resulted in the induction of both tumor-specific and virus-specific immunity as measured by ELISPOT and pentamer analysis. This is the first time that oncolytic virus–mediated antitumor immunity has been shown in humans. Ad5-D24-GMCSF is promising for further clinical testing. Cancer Res; 70(11); 4297–309. ©2010 AACR.

PDF file - 82K, Figure S1. Expression analysis of Chk1 and CIP2A in human malignancies.

PDF file - 123K, Figure S3. Effect of cell cycle on CIP2A expression.

PDF file - 90K, Supplementary Table S2. List of genes which expression significantly correlates with Chk1 and CIP2A.

PDF file - 205K

PDF file - 101K, Figure S4. In vitro and in vivo regulation of CIP2A mRNA expression by Chk1 and effect on cancer cell viability and anchorage independent growth by Chk1, Claspin and CIP2A.

Checkpoint kinase Chk1 is constitutively active in many cancer cell types and new generation Chk1 inhibitors show marked antitumor activity as single agents. Here we present a hitherto unrecognized mechanism that contributes to the response of cancer cells to Chk1-targeted therapy. Inhibiting chronic Chk1 activity in cancer cells induced the tumor suppressor activity of protein phosphatase protein phosphatase 2A (PP2A), which by dephosphorylating MYC serine 62, inhibited MYC activity and impaired cancer cell survival. Mechanistic investigations revealed that Chk1 inhibition activated PP2A by decreasing the transcription of cancerous inhibitor of PP2A (CIP2A), a chief inhibitor of PP2A activity. Inhibition of cancer cell clonogenicity by Chk1 inhibition could be rescued in vitro either by exogenous expression of CIP2A or by blocking the CIP2A-regulated PP2A complex. Chk1-mediated CIP2A regulation was extended in tumor models dependent on either Chk1 or CIP2A. The clinical relevance of CIP2A as a Chk1 effector protein was validated in several human cancer types, including neuroblastoma, where CIP2A was identified as an NMYC-independent prognostic factor. Because the Chk1–CIP2A–PP2A pathway is driven by DNA-PK activity, functioning regardless of p53 or ATM/ATR status, our results offer explanative power for understanding how Chk1 inhibitors mediate single-agent anticancer efficacy. Furthermore, they define CIP2A-PP2A status in cancer cells as a pharmacodynamic marker for their response to Chk1-targeted therapy. Cancer Res; 73(22); 6757–69. ©2013 AACR.

Lynch syndrome (LS) is a hereditary cancer predisposition syndrome caused by germline mutations in the DNA mismatch repair (MMR) genes resulting in an increased risk of colorectal cancer (CRC) and other malignancies. Those germline defects with other genetic and epigenetic changes accelerate multistep tumorigenesis. However, early events leading to polyp formation and the timing and order of the molecular “hits” remain unknown. Studies on sporadic CRC have indicated that promoter hypermethylation leading to gene silencing can act as an alternative mechanism to mutations at early stages of tumorigenesis, but its importance in hereditary CRC is obscure. In this study we aimed to define 1) methylation changes that occur at different stages of LS-associated colorectal tumor progression, and 2) somatic mutations that occur in adenomas comparing the status of low- and high-grade dysplasia and MMR deficiency (MMR-D) and proficiency (MMR-P). Colorectal biopsies including normal mucosae, adenomas and carcinomas were prospectively collected from 104 LS patients during colonoscopy surveillance, supplemented with retrospective tumor specimens from 56 patients. Promoter methylation was analyzed using the methylation-specific multiplex ligation-dependent probe amplification test (MS-MLPA) including selected tumor suppressor genes (TSGs) associated with early colon oncogenesis. Hypermethylation tendency was evaluated based on the frequency of CpG island methylator phenotype (CIMP) according to the methylation status of eight established CIMP marker genes. LINE-1 methylation was studied as a surrogate marker for global hypomethylation. Immunohistochemistry was used to detect MMR protein expression in neoplastic lesions. As an ongoing study mutation status of adenomas are studied using the Ion AmpliSeqTM Colon and Lung Cancer Panel targeting mutational hot spots in 22 genes. Results indicate that the expression of the MMR protein corresponding to the gene mutated in the germline decreases along with dysplasia but occurs as a relatively late event in the tumor progression, suggesting the presence of other somatic events that drive neoplastic transformation. Taken together, methylation of TSGs and frequency of CIMP increased and LINE-1 methylation decreased in adenomas and carcinomas along with dysplasia. However, a significant increase of methylation in some genes was detected already in adenomas with low-grade dysplasia compared to normal mucosae. In addition, a proportion of low-grade adenomas could already be classified CIMP positive. When comparing MMR-P and MMR-D adenomas, LINE-1 methylation decreased along the loss of MMR protein expression, and interestingly, higher methylation of SFRP1 was observed in MMR-P adenomas compared to MMR-D cases and normal mucosae. These findings emphasize the importance and early appearance of epigenetic changes in LS-associated tumorigenesis. Citation Format: Satu Mäki-Nevala, Satu Valo, Ari Ristimäki, Laura Renkonen-Sinisalo, Anna Lepistö, Jukka-Pekka Mecklin, Päivi Peltomäki. Methylation changes and somatic mutations as early events in Lynch syndrome-associated colorectal cancer [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 3081.

Small bowel adenocarcinoma (SBA) is a rare but aggressive cancer type with limited treatment options. Known predisposing factors include Crohn's disease, celiac disease, and hereditary syndromes such as familial adenomatous polyposis (FAP), Lynch syndrome, and Peutz-Jeghers syndrome. Here, our aim was to further characterize genetic susceptibility to SBA in a large population-based cohort and simultaneously demonstrate the ability to utilize tumor-only data to cost-effectively but reliably call germline variants. Information on all SBAs diagnosed in Finland between the years 2003-2011 were collected utilizing the Finnish Cancer Registry that maintains a nation-wide database on all cancer cases diagnosed in Finland since 1953. From these we selected all SBAs 1) confirmed as small bowel primary tumor, 2) with available tumor material, and 3) tumor content of at least 50%. Additionally, all relevant medical records were available for all cases. Altogether 106 tumors representing all three parts of the small bowel were selected for exome sequencing. The variant calls were produced with GATK HaplotypeCaller. Germline calls were extracted from the data by filtering out somatic calls that were originally produced by e.g. filtering SNV and indel calls against whole-genome and exome samples of the GnomAD dataset (n=138,632). To focus on possible disease-causing variants, the remaining putative germline variants with allele frequency >0.001 in the whole GnomAD and population-specific Finnish GnomAD set (n=1,747) were excluded. The germline origin of the observed, most prominent variants are being verified by Sanger sequencing, whenever corresponding normal DNA is available. First, we considered variants that were truncating or predicted damaging in silico in the 106 known cancer predisposing genes according to COSMIC. Eight of the 106 genes harbored such variants in at least two patients. We detected the pathogenic germline variant in all patients known to have a hereditary cancer syndrome (MLH1/MSH6 in three Lynch syndrome and APC in two FAP cases). We also identified two patients with a BRCA2 germline variant, one truncating and the other one predicted damaging. BRCA2 might play a role in SBA, thus far germline BRCA2 variants have been observed at least in cancers of the ampullary region. Next, we widened the analysis for other candidate genes but preliminary results show no clear candidates that would be shared by several patients. We will also look more closely into genes belonging to the same signaling pathways as the known syndrome causing genes. Finally, no single gene mutated in all the patients with celiac disease (n=10) was observed nor were there clear differences in the germlines of patients with and without celiac disease. This population-based study on predisposing variants in SBAs provides new information on their molecular genetic background, possibly having an impact also on their treatment. Citation Format: Ulrika A. Hänninen, Riku Katainen, Tomas Tanskanen, Jiri Hamberg, Ari Ristimäki, Eero Pukkala, Minna Taipale, Jukka-Pekka Mecklin, Mervi Aavikko, Linda M. Forsström, Esa Pitkänen, Netta Mäkinen, Lauri A. Aaltonen. Identification of predisposing genes for small bowel adenocarcinoma by exome sequencing [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-382.

It has been proposed that human tumors contain stem cells that have a central role in tumor initiation and posttreatment relapse. Putative breast cancer stem cells may reside in the CD44+CD24−/low population. Oncolytic adenoviruses are attractive for killing of these cells because they enter through infection and are therefore not susceptible to active and passive mechanisms that render stem cells resistant to many drugs. Although adenoviruses have been quite safe in cancer trials, preclinical work suggests that toxicity may eventually be possible with more active agents. Therefore, restriction of virus replication to target tissues with tissues-specific promoters is appealing for improving safety and can be achieved without loss of efficacy. We extracted CD44+CD24−/low cells from pleural effusions of breast cancer patients and found that modification of adenovirus type 5 tropism with the serotype 3 knob increased gene delivery to CD44+CD24−/low cells. α-Lactalbumin, cyclo-oxygenase 2, telomerase, and multidrug resistance protein promoters were studied for activity in CD44+CD24−/low cells, and a panel of oncolytic viruses was subsequently constructed. Each virus featured 5/3 chimerism of the fiber and a promoter controlling expression of E1A, which was also deleted in the Rb binding domain for additional tumor selectivity. Cell killing assays identified Ad5/3-cox2L-d24 and Ad5/3-mdr-d24 as the most active agents, and these viruses were able to completely eradicate CD44+CD24−/low cells in vitro. In vivo, these viruses had significant antitumor activity in CD44+CD24−/low–derived tumors. These findings may have relevance for elimination of cancer stem cells in humans. [Cancer Res 2008;68(14):5533–9]

HuR is a ubiquitously expressed mRNA-binding protein. Intracellular localization of HuR is predominantly nuclear, but it shuttles between the nucleus and the cytoplasm. In the cytoplasm it can stabilize certain transcripts. Because nucleocytoplasmic translocation of HuR is necessary for its activity, it was hypothesized that cytoplasmic HuR expression in cancer cells could be a prognostic marker. To test the significance of HuR in carcinogenesis of the breast, we have investigated HuR expression in a mouse mammary gland tumor model and from 133 invasive ductal breast carcinoma specimens. HuR expression was elevated in the cyclooxygenase-2 transgene–induced mouse mammary tumors, and its expression was predominantly cytoplasmic in the tumor cells. In the human carcinoma samples, high cytoplasmic immunoreactivity for HuR was found in 29% (38 of 133) of the cases. Cytoplasmic HuR expression associated with high grade (P = 0.0050) and tumor size over 2 cm (P = 0.0082). Five-year distant disease-free survival rate was 42% [95% confidence interval (95% CI), 26-58] in cytoplasm-high category and 84% (95% CI, 76-91) in cytoplasm-negative or -low category (P < 0.0001), and high cytoplasmic expression of HuR was an independent prognostic factor in a Cox multivariate model (relative risk 2.07; 95% CI, 1.05-4.07). Moreover, high cytoplasmic HuR immunopositivity was significantly associated with poor outcome in the subgroup of node-negative breast cancer in a univariate analysis (P < 0.0007). Our results show that high cytoplasmic HuR expression is associated with a poor histologic differentiation, large tumor size, and poor survival in ductal breast carcinoma. Thus, HuR is the first mRNA stability protein of which expression associates with poor outcome in breast cancer.

Colorectal cancer (CRC) develops via multiple steps which involve genetic changes, such as mutations in growth-regulatory genes, and epigenetic alterations, such as CpG island hypermethylation. Lynch syndrome (LS) is one of the most common inherited cancer susceptibility syndromes and is associated with inherited defects of the DNA mismatch repair (MMR) genes, which together with other genetic and epigenetic changes are known to accelerate tumorigenesis. In this investigation we explored the timing of CpG island methylation changes in the course of transformation of normal colonic mucosa to pre-cancerous and cancerous lesions in genetically predisposed individuals. Colorectal neoplastic lesions including low-grade adenomas, high-grade adenomas and carcinomas together with matching normal tissue were gathered from 43 LS mutation carriers enrolled in colonoscopy surveillance. Frequency of CpG island methylator phenotype (CIMP) in different tumor types was analyzed with the methylation-specific multiplex ligation-dependent probe amplification test (MS-MLPA) which detects simultaneously the methylation status of eight CIMP marker genes. Additionally, we analyzed the methylation status of seven selected tumor suppressor genes previously associated with early colon oncogenesis. Immunohistochemistry was used to detect MMR protein expression in neoplastic lesions. We show that the expression of the MMR protein corresponding to the germline mutation decreases along with increasing dysplasia in tumors. Similarly, methylation increases in LS adenomas and carcinomas together with dysplasia grade in comparison to normal colonic mucosa. A proportion of low-grade adenomas could already be classified CIMP positive, and the frequency of CIMP further increased in high-grade adenomas and carcinomas. Our results offer new insights into the molecular mechanisms through which DNA methylation can affect colorectal carcinogenesis in LS mutation carriers. Citation Format: Satu Valo, Sippy Kaur, Ari Ristimäki, Laura Renkonen-Sinisalo, Heikki Järvinen, Jukka-Pekka Mecklin, Minna Nyström, Päivi Peltomäki. DNA methylation changes in Lynch syndrome associated colorectal adenomas and carcinomas. [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 4766. doi:10.1158/1538-7445.AM2015-4766

Small intestinal neuroendocrine tumors (SI-NETs) originating from the enterochromaffin cells of the intestinal mucosa are among the most common tumors in the small intestine. In the past few decades the incidence of SI-NETs has increased more than four-fold in the Western countries. Although SI-NETs are usually well differentiated and indolent with low mitotic activity, multiple discontinuous tumors are present in up to 1/3 of the patients and metastases are found in the majority of the patients at diagnosis. Previous studies have described significantly elevated risk for gastrointestinal NETs in family members of affected individuals. Our recent nationwide registry-based analyses also showed high familial enrichment for SI-NETs. MEN1 syndrome (MIM#131100) patients are often affected by foregut NETs, but no genetic predisposition factor is known for SI-NETs. We have studied a Finnish family of five affected individuals with multiple SI-NETs. The affected individuals had also been diagnosed with numerous adenomas of the colon. The family exhibits autosomal dominant inheritance of SI-NETs with affected individuals in two generations. To identify the possible genetic predisposing factor, we performed genome-wide SNP genotyping and linkage analysis followed by exome and genome sequencing. In addition, we have studied the somatic alterations of the tumors by genome-wide copy number analysis and exome sequencing. We have identified candidate chromosomal regions and genetic variants in this family. Currently we are functionally validating the pathogenicity of the variants and screening the variants in additional familial and sporadic cases with SI-NETs, including a Swedish family of three affected individuals in three generations and Finnish familial and sporadic cases identified through Finnish Cancer Registry. Increased understanding of tumor susceptibility is of great importance in creating tools for better diagnosis and management of the patients. Characterization of novel tumor susceptibility conditions and identification of the associated gene defects also enable studies on gene's biological function and role in other relevant phenotypes or associated tumor types. Citation Format: Mervi Aavikko, Eevi Kaasinen, Iikki Donner, Kaisa Lehti, Erika Gucciardo, Bideep Shrestha, Jukka-Pekka Mecklin, Järnhult Johannes, Kalle Landerholm, Eero Pukkala, Camilla Schalin-Jäntti, Ari Ristimäki, Pia Vahteristo, Lauri A. Aaltonen. Familial multiple metastatic small intestine neuroendocrine tumors: searching for genetic susceptibility. [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 2744. doi:10.1158/1538-7445.AM2015-2744

Colorectal cancer (CRC) develops via multiple steps which involve genetic changes, such as mutations in growth-regulatory genes, and epigenetic alterations, such as CpG island hypermethylation. These changes accumulate over time in the normal tissue, for example due to many environmental factors such as diet, and may result in pre-cancerous field defects. Genes having high level of methylation in peritumoral tissues could be used as biomarkers for colorectal carcinogenesis. The most common familial cancer susceptibility syndromes are familial adenomatous polyposis (FAP), caused by inherited mutations in the APC tumor suppressor gene, and Lynch syndrome (LS), associated with inherited defects of the DNA mismatch repair (MMR) genes which together with other genetic and epigenetic changes are known to accelerate tumorigenesis. This investigation was designed to define how CpG island methylation of CRC risk genes varies in pre-cancerous and cancerous lesions, adjacent normal mucosa and distinct normal mucosa from genetically predisposed individuals. Colonic mucosa biopsies from different colorectal regions were gathered from 40 Finnish LS mutation carriers and 23 FAP patients during regular surveillance. In addition with normal mucosa biopsies, all adenoma and carcinoma findings from these individuals together with matching normals were gathered and analyzed. Patient information was obtained from the nation-wide registry. CpG island methylation was studied with the methylation-specific multiplex ligation-dependent probe amplification test (MS-MLPA) which detects CpG methylation of selected genes. Immunohistochemistry was used to detect MMR protein expression in neoplastic lesions. Of 16 CRC risk genes tested so far 9 show increased methylation in polyps/adenomas/carcinomas. Mean methylation values increase along with age in both syndromes, with polyps from LS showing the highest proportional increase relative to normal tissues. The results offer new insights to the molecular mechanisms through which DNA methylation can affect colorectal carcinogenesis in LS and FAP mutation carriers. Citation Format: Satu Valo, Sippy Kaur, Ari Ristimäki, Laura Renkonen-Sinisalo, Heikki Järvinen, Jukka-Pekka Mecklin, Minna Nyström, Päivi Peltomäki. DNA methylation changes in Lynch syndrome and FAP-associated colorectal tumorigenesis. [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 417. doi:10.1158/1538-7445.AM2014-417

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

DNA damage is a hallmark of malignantly transformed cells. Accordingly, overexpression of the DNA damage activated kinases has been observed in different human cancers. However, it is unclear whether constitutive DNA damage present in unperturbed cancer cells promotes tumourigenesis. Here we show that increased activity of DNA damage sensitive kinase Chk1 promotes proliferation of several cancer cell types by stimulating expression of CIP2A protein. Either chemical or genetic inhibition of Chk1, and not of ATM-ATR, results in potent inhibition of CIP2A protein expression levels. Importantly, Chk1 siRNA-elicited inhibition of cell proliferation and clonogenic growth is rescued by overexpression of CIP2A from a heterologous promoter. Moreover, in clinical (human) tumor samples there is a significant association between CIP2A and Chk1 expression in both ovarian and gastric cancers. Intriguingly, meta-analysis of seventeen published genome wide studies on different types of cancers unveils a striking similarity in the gene expression patterns of both CIP2A and Chk1. Amongst these, 12/17 studies show overexpression (top 10%) of both these proteins. Additionally, similarly to CIP2A, increased Chk1 expression is shown to correlate with tumor progression in human cancer. In summary, these results identify a novel function for DNA damage kinase Chk1 in regulation of the human oncoprotein CIP2A. In general, these results provide an unprecented molecular mechanism by which constitutive DNA damage present in cancer cells promotes tumourigenesis. Additionally, as CIP2A is not significantly expressed in most of the normal human tissues, it is proposed that targeting of CIP2A might abrogate the Chk1-mediated support of proliferation without concerns related to anticancer therapy directly targeting Chk1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4194. doi:10.1158/1538-7445.AM2011-4194

Cancerous inhibitor of PP2A (CIP2A) is a recently identified oncoprotein, but its role in ovarian cancer is not known. We analyzed CIP2A protein expression in 562 consecutive serous ovarian cancer patients, of which 524 were scored successfully and analyzed for presence of CIP2A immunopositivity using tissue microarrays. The association of CIP2A expression with survival was evaluated according to the Kaplan-Meier method. We found strong CIP2A positive immunostaining in 212 (40.4%) specimens, 222 (42.4%) were weakly positive, while the rest (n = 90, 17.2%) were immunonegative for CIP2A protein. Our results demonstrate that CIP2A immunopositivity is a marker of reduced overall survival in ovarian cancer patients (P < 0.0001). In addition, positive CIP2A expression was significantly more frequent in specimens with high grade (P < 0.0001), advanced stage (P = 0.0005), aberrant p53 immunoreactivity (P < 0.0001), high proliferation index (P < 0.0001), and aneuploidy (P = 0.001). Interestingly, in subgroups of patients with favorable clinical factors (i.e. low stage and optimal debulking surgery) CIP2A expression was strongly associated with reduced survival (P < 0.0001 for both parameters). This shows that CIP2A could be used to predict biological behavior in the group of patients with otherwise favorable prognosis. In conclusion, our results show that CIP2A protein expression is a novel marker of reduced survival in patients with serous ovarian carcinoma. Furthermore, these results suggest that CIP2A characterizes the aggressive type of this disease even within subgroups with initially favorable prognosis. Citation 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 4645.

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.