Loss Of Tumor Suppressor Genes Research Articles

Abstract BS02-2: Alterations in tumor metabolism to support breast tumor growth and progression

Abstract Breast cancer cells must adapt metabolism to support anabolic processes and allow the accumulation of biomass to allow proliferation. However, tumor cells also experience periods of stress, and metabolic plasticity to shift metabolism to maximize efficiency and survive nutrient limitation is also needed for cancer progression. The M2 pyruvate kinase isoform (PKM2) is expressed in cancers and plays a role in regulating metabolism. To determine whether PKM2 is required for the development and growth of primary breast tumors, we generated mice harboring a conditional allele for PKM2. PKM2 deletion selectively abolishes PKM2 protein production while continued production of PKM1, the other major pyruvate kinase isoform. We crossed PKM2 conditional mice to a model of human breast cancer driven by loss of the Brca1 tumor suppressor gene. Contrary to prior expectation, mammary tumor formation and progression was accelerated despite PKM2 deletion. PKM2-null tumors displayed heterogeneous compensatory PKM1 expression, and expression of PKM1 was observed only in non-proliferating tumor cells. In contrast, proliferating cells in the tumor had no detectable pyruvate kinase expression. This suggests that PKM2 protein is not necessary for tumor cell proliferation and implies that the inactive state of PKM2 is associated with the proliferating cell population within tumors, while cells with active pyruvate kinase are involved in aspects of tumor progression that do not involve proliferation. Consistent with these findings, variable expression of PKM2 was observed across a panel of human breast tumors, and recurrent mutations resulting in disruption of one PKM2 allele are found in multiple human cancers. Together, these data support a model where regulation of PKM2 activity allows the enzyme to support the differential metabolic requirements of both proliferating and non-proliferating tumor cells. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr BS02-2.

Loss of LKB1 and PTEN tumor suppressor genes in the ovarian surface epithelium induces papillary serous ovarian cancer

Epithelial ovarian cancer presents mostly with serous, endometrioid or mucinous histology but is treated as a single disease. The development of histotype-specific therapy has been challenging because of the relative lack of studies attributing disrupted pathways to a distinct histotype differentiation. mTOR activation is frequently associated with poor prognosis in serous ovarian cancer, which is the most common and most deadly histotype. However, the mechanisms dysregulating mTOR in the pathogenesis of ovarian cancer are unknown. We detected copy number loss and correlated lower expression levels of LKB1, TSC1, TSC2 and PTEN tumor suppressor genes for upstream regulators of mTOR activity in up to 80% in primary ovarian serous tumor databases, with LKB1 allelic loss-predominant. Reduced LKB1 protein was usually associated with increased mTOR activity in both serous ovarian cancer cell lines and primary tumors. Conditional deletion of Lkb1 in murine ovarian surface epithelial (OSE) cells caused papillary hyperplasia and shedding but not tumors. Simultaneous deletion of Lkb1 and Pten, however, led to development of high-grade ovarian serous histotype tumors with 100% penetrance that expressed WT1, ERα, PAX8, TP53 and cytokeratin 8, typical markers used in the differential diagnosis of serous ovarian cancer. Neither hysterectomy nor salpingectomy interfered with progression of ovarian tumorigenesis, suggesting that neither uterine nor Fallopian tube epithelial cells were contributing to tumorigenesis. These results implicate LKB1 loss in the OSE in the pathogenesis of serous ovarian cancer and provide a compelling rationale for investigating the therapeutic potential of targeting LKB1 signaling in patients with this deadly disease.

Mouse model of intrahepatic cholangiocarcinoma validates FIG–ROS as a potent fusion oncogene and therapeutic target

Cholangiocarcinoma is the second most common primary liver cancer and responds poorly to existing therapies. Intrahepatic cholangiocarcinoma (ICC) likely originates from the biliary tree and develops within the hepatic parenchyma. We have generated a flexible orthotopic allograft mouse model of ICC that incorporates common genetic alterations identified in human ICC and histologically resembles the human disease. We examined the utility of this model to validate driver alterations in ICC and tested their suitability as therapeutic targets. Specifically, we showed that the fused-in-glioblastoma-c-ros-oncogene1 (FIG-ROS1(S); FIG-ROS) fusion gene dramatically accelerates ICC development and that its inactivation in established tumors has a potent antitumor effect. Our studies establish a versatile model of ICC that will be a useful preclinical tool and validate ROS1 fusions as potent oncoproteins and therapeutic targets in ICC and potentially other tumor types.

Complementation of non-tumorigenicity of HPV18-positive cervical carcinoma cells involves differential mRNA expression of cellular genes including potential tumor suppressor genes on chromosome 11q13

The fusion between human tumorigenic cells and normal human diploid fibroblasts results in non-tumorigenic hybrid cells, suggesting a dominant role for tumor suppressor genes in the generated hybrid cells. After long-term cultivation in vitro, tumorigenic segregants may arise. The loss of tumor suppressor genes on chromosome 11q13 has been postulated to be involved in the induction of the tumorigenic phenotype of human papillomavirus (HPV)18-positive cervical carcinoma cells and their derived tumorigenic hybrid cells after subcutaneous injection in immunocompromised mice. The aim of this study was the identification of novel cellular genes that may contribute to the suppression of the tumorigenic phenotype of non-tumorigenic hybrid cells in vivo. We used cDNA microarray technology to identify differentially expressed cellular genes in tumorigenic HPV18-positive hybrid and parental HeLa cells compared to non-tumorigenic HPV18-positive hybrid cells. We detected several as yet unknown cellular genes that play a role in cell differentiation, cell cycle progression, cell-cell communication, metastasis formation, angiogenesis, antigen presentation, and immune response. Apart from the known differentially expressed genes on 11q13 (e.g., phosphofurin acidic cluster sorting protein 1 (PACS1) and FOS ligand 1 (FOSL1 or Fra-1)), we detected novel differentially expressed cellular genes located within the tumor suppressor gene region (e.g., EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) and leucine rich repeat containing 32 (LRRC32) (also known as glycoprotein-A repetitions predominant (GARP)) that may have potential tumor suppressor functions in this model system of non-tumorigenic and tumorigenic HeLa x fibroblast hybrid cells.

Integrative Genomic Characterization of Oral Squamous Cell Carcinoma Identifies Frequent Somatic Drivers

The survival of patients with oral squamous cell carcinoma (OSCC) has not changed significantly in several decades, leading clinicians and investigators to search for promising molecular targets. To this end, we conducted comprehensive genomic analysis of gene expression, copy number, methylation, and point mutations in OSCC. Integrated analysis revealed more somatic events than previously reported, identifying four major driver pathways (mitogenic signaling, Notch, cell cycle, and TP53) and two additional key genes (FAT1, CASP8). The Notch pathway was defective in 66% of patients, and in follow-up studies of mechanism, functional NOTCH1 signaling inhibited proliferation of OSCC cell lines. Frequent mutation of caspase-8 (CASP8) defines a new molecular subtype of OSCC with few copy number changes. Although genomic alterations are dominated by loss of tumor suppressor genes, 80% of patients harbored at least one genomic alteration in a targetable gene, suggesting that novel approaches to treatment may be possible for this debilitating subset of head and neck cancers.

Perturbation of nucleotide metabolism - the driving force of oncogene-induced senescence

Induction of premature (stress induced) cellular senescence of normal cells provides the mechanism eliminating proliferative capability of potentially carcinogenic cells and thereby is considered to be a barrier preventing neoplastic tranformation [1]. On the other hand, induction of senescence in tumor cells provides a therapeutic opportunity to treat cancers that may be potentially susceptible [2]. Among factors inducing senescence are oxidative DNA damage leading to persistent replication stress, activation of oncogenes and loss of tumor suppressor genes [1]. It was recently reported however that perturbation of the nucleotide metabolism can be added into these already “classic” inducers. The early observations by Mannava et al.,[3]and Liu et al [4], established evidence of a cause-effect relationship between C-MYC, the oncogene that is capable of suppressing senescence and nucleotide metabolism. The authors reported that downregulation of the C-MYC in several human metastatic melanoma lines via lentivirus-based shRNA led to repression of several genes encoding enzymes related to dNTP metabolism such as thymidylate synthase (TS), inosine monophosphate dehydrogenase 2 (IMPDH2) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2). This observation provided a rationale to explore a potential link between dNTP metabolism, DNA damage and the oncogene induced senescence (OIS). Additional evidence on the role of perturbed dNTP pools in induction of DNA damage and replication stress was provided by Bester et al.,[5]. Their studies were focused on oncogenes regulating cell cycle progression, specifically the Rb-E2F pathway. The authors observed that aberrant activation of this pathway by HPV-16 E6/E7 or cyclin E oncogenes led to a reduction of cellular nucleotide levels. Interestingly, when C-MYC was coexpressed with cyclin E, its presence led to an increase in transcription of nucleotide biosynthesis genes, prevented the reduction of nucleotide pool and rescued the replication-stress induced DNA damage [5]. In the subsequent studies Mannava et al.,[6] provided direct evidence that down-regulation of nucleoside pools is a cause of DNA damage and senescence in normal human fibroblasts (NHFs) overexpressing HRASG12V, a classical model of oncogene-induced senescence. Among the enzymes down-regulated by activated HRAS were TS, ribonucleotide reductase subunits M1 and M2 (RRM1 and RRM2). Ectopic coexpression of TS, RRM1 and RRM2 or addition of deoxyribonucleosides to a large extent prevented DNA damage, decreased expression of senescence-associated phenotypes as well as attenuated the extent of proliferation arrest. Interestingly, overexpression of TS, RRM1 and RRM2 in quiescent NHFs had no effect on their proliferation arrest suggesting that unlike the OIS, quiescence is not induced by perturbation of dNTP pools. Moreover, in a separate study, Mannava et al.,[7]demonstrated that shRNA-mediated depletion of RRM2 and TS was sufficient to induce senescence in melanoma cells. The observation that knockdown of RRM2 and TS in cells with ctivated oncogenes drives them to senescence provides further evidence that abundance of dNTPs promotes melanoma by suppressing cellular senescence. Most of the above findings were reproduced in a study of Aird et al.,[8] on a role of suppressed nucleotide pools in DNA damage and oncogene-induced senescence. Additionally the authors reported that RRM2 repression correlated with senescent status of melanocytes in human benign nevi and melanoma harboring oncogenic BRAF or NRAS. Furthermore, high expression of RRM2 correlates with poor survival of melanoma patiens[8]. The seminal findings on different cellular and oncogene models that perturbation of dNTP pools leads to DNA damage, replication stress, irreversible senescence [5-8],may have important clinical implications. The example that with similar oncogene activation the senescence of melanocytes induced by shortage of dNTPs results in nevi remaining benign for years whereas abundance of dNTPs leads to malignant melanoma, opens new possibilities to explore potential role of dNTPs as the target in cancer treatment. In light of these findings the commonly used anticancer drug hydroxyurea which targets dNTPs or 5-fluorouracil that affects thymidylates should be revaluated in terms of its possible senescence-promoting efficiency. For years, acceptance of antitumor drugs by the FDA was based on response criteria assessed by tumor shrinkage that most likely occurs as a result of rapid cell death by apoptosis. Assessment of the tumor response by its size reduction criteria precluded the detection of potential treatment benefits that could be mediated by induction of senescence of tumor cells which may remain alive but reproductively dead. It is tempting to explore whether a combined treatment of cancer initially by cytotoxic regimen, relying on induction of apoptosis, be followed by prolonged treatment promoting senescence of cancer cells, is more effective than the traditional chemotherapy.

Integrated high-resolution array CGH and SKY analysis of homozygous deletions and other genomic alterations present in malignant mesothelioma cell lines

High-resolution oligonucleotide array comparative genomic hybridization (aCGH) and spectral karyotyping (SKY) were applied to a panel of malignant mesothelioma (MMt) cell lines. SKY has not been applied to MMt before, and complete karyotypes are reported based on the integration of SKY and aCGH results. A whole genome search for homozygous deletions (HDs) produced the largest set of recurrent and non-recurrent HDs for MMt (52 recurrent HDs in 10 genomic regions; 36 non-recurrent HDs). For the first time, LINGO2, RBFOX1/A2BP1, RPL29, DUSP7, and CCSER1/FAM190A were found to be homozygously deleted in MMt, and some of these genes could be new tumor suppressor genes for MMt. Integration of SKY and aCGH data allowed reconstruction of chromosomal rearrangements that led to the formation of HDs. Our data imply that only with acquisition of structural and/or numerical karyotypic instability can MMt cells attain a complete loss of tumor suppressor genes located in 9p21.3, which is the most frequently homozygously deleted region. Tetraploidization is a late event in the karyotypic progression of MMt cells, after HDs in the 9p21.3 region have already been acquired.

Abstract 4208: Integrated oligo aCGH and SKY analysis of genomic alterations in malignant mesothelioma cell lines.

Abstract Malignant mesothelioma (MMt) is a rare and very aggressive tumor of the mesothelium. It's most prevalent subtype is malignant pleural mesothelioma, eighty percent of which are associated with asbestos exposure. MMt incidence is 0.9 new cases per 100,000 persons per year. The five year relative survival is 7% overall, which positions MMt immediately above pancreatic cancer (6%), at the bottom of the survival list by cancer site. We applied a comprehensive molecular cytogenetic analysis using spectral karyotyping (SKY) and oligo aCGH to a panel of MMt cell lines. We found the largest set of recurrent and non-recurrent homozygous deletions (HD) for MMt (88 HDs in 17 cell lines; 52 recurrent HDs that fall into ten genomic areas). Detailed analysis of the most frequently homozygously deleted area located at 9p21.3 revealed that the main targets are CDKN2A/p14ARF (in 100% of the MM cell lines), CDKN2A/p16INK4A and CDKN2B /p15 (94% of cell lines). p14ARF seems to be the main target of HDs in the 9p21.3 area (deleted in all 17 MMt cell lines studied), followed by p16INK4A (deleted in 16 cell lines) indicating the inactivation of two major tumor suppressing pathways, p53 and Rb. Accordingly, mutations or HDs of p53 and Rb are infrequent events in MMt tumors. Other recurrent HDs were located at 9p21.2 (targeting LINGO2) and at16p13.3 (RBFOX1) in 41% of cell lines, 22q11.23 (GSTT1) in 29%, 22q12.2 (NF2) and 3q26 (no known genes) in 23% each, 8p11.22 -23 (ADAM5P/ADAM3A) in 18%, 3p21.3-p21.2 (RPL29), 3p21 (DUSP7), 4q22.1 (FAM190A) and 13q11-q12 (LATS2) in 12% each of the 17 MM cell lines While p16INK4A, p14ARF, p15, LATS2 and NF2 have been shown to behave as tumor suppressors in MMts, other genes in the HD areas presented here could be new tumor suppressor candidates. Structural chromosomal rearrangements were mainly non-reciprocal translocations (74% of aberrations detected by SKY) and deletions (23%). No recurrent balanced or unbalanced translocations have been found. Structural chromosomal rearrangements resulted in recurrent losses of fragments or whole chromosomal arms of 1p, 3p, 8p, 9p, 13q, 14q, 15q, 18q, 22q and gains of 1q, 5p, 7p, 8q, 17q, 20q. Small HDs were frequently embedded in the areas of focal losses. Integration of SKY and aCGH data allowed reconstruction of chromosomal rearrangements leading to the formation of HDs. Our data imply that only with acquisition of structural or numerical karyotypic abnormalities can MMt cells attain a complete loss of the 9p21.3 genomic area and loss of tumor suppressor genes located there. Tetraploidization seems to be a late event in the karyotypic progression of MMt cells, after HD in the 9p21.3 area has already been acquired. Citation Format: Ester Rozenblum, Geula Klorin, Oleg Glebov, Robert L. Walker, Yoonsoo Park, Paul S. Meltzer, Ilan R. Kirsch, Frederic J. Kaye, Anna V. Roschke. Integrated oligo aCGH and SKY analysis of genomic alterations in malignant mesothelioma cell lines. [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 4208. doi:10.1158/1538-7445.AM2013-4208

Abstract 4251: Genomewide methylation and expression profiles in cervical cancer.

Abstract Cervical carcinoma is one of the most common cancers in females. This cancer begins with precancerous stages and can evolves to carcinoma over 10 years or more. Cervical cancer is a multistep process with accumulation of genetic and epigenetic alterations in regulatory genes, leading to activation of oncogenes and inactivation or loss of tumor suppressor genes. We aimed to identify genetic alterations associated with pre-cancerous stage and also with cervical cancer. For this purpose, microarray comparative genomic hybridization(Illumina 450K and Illumina Expression HT12 Human CGH Whole-Genome Arrays) was performed with 16 cases of cervical intraepithelial neoplasia(CIN) of different stages and 3 cases of carcinoma. We identified epigenetic inactivation of TSGs by hyper- or hypo-methylation which has been recognized as an important and alternative mechanism in tumorigenesis. Here we discuss these epigenetic alterations in cervical intrapeithelial neoplasia and cervical cancer focusing on DNA methylation. Citation Format: Kwon Yongil, Nakwoo Lee, Sungjong Lee. Genomewide methylation and expression profiles in cervical 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 4251. doi:10.1158/1538-7445.AM2013-4251

Abstract 3147: Cancer-specific mutations in adult soft tissue sarcomas.

Abstract Understanding of how cancer initiates and progresses is a fundamental goal of cancer research. Identification of cancer mutations is of profound importance for both understanding of the disease and for developing novel therapies. Soft-tissue sarcomas include multiple uncommon histotypes with features of diverse connective tissue lineages. Although some of their underlying genetic changes have been elucidated, for example in those types which contain etiologic chromosome translocations, in general, the genetic basis of these tumors is incompletely described. To investigate mutations in 8 subtypes of adult soft tissue sarcomas, we resequenced coding exons from about 1300 cancer relevant genes in 66 tumor samples. Data was analyzed for somatic variants in samples with matched normals while samples without normal were analyzed individually. The most frequent events were TP53 mutations (26%) followed by RB1 (9%) and APC (8%) mutations. Even though, well-defined oncogene activating mutations were present, they were very rare. Given the prevalence of tumor suppressor mutations in these samples we are currently integrating CGH results to look for chromosomal deletions to support loss of tumor suppressor genes. In addition, we are investigating low-frequency mutations in lesser-known genes that may be of potential interest. Our preliminary results suggest that these subtypes of soft-tissue sarcomas depend on tumor-suppressor loss predominantly, and may have gained multiple complex sets of mutations in a number of other pathways. In conclusion, our work will be important in cataloging novel cancer mutations in rare types of adult sarcomas, with potential implications for understanding their biology and the development of new therapies. Citation Format: Ogan D. Abaan, Princy Francis, Marbin Pineda, Robert L. Walker, Yuelin J. Zhu, Sven Bilke, Sean R. Davis, Paul S. Meltzer. Cancer-specific mutations in adult soft tissue sarcomas. [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 3147. doi:10.1158/1538-7445.AM2013-3147

Abstract 351: Gliomagenesis via the reactivation of quiescent adult oligodendrocyte precursor cells.

Abstract Malignant gliomas are devastating, incurable brain tumors. Surprisingly, though the proliferative activity of brain cells decreases precipitously with age, glioma occurs most frequently in the elderly. Our lab recently showed that introducing p53 and NF1 mutations into perinatal oligodendrocyte precursor cells (OPCs) can lead to gliomagenesis. However, perinatal OPCs are highly migratory and proliferative, whereas adult OPCs are much more quiescent. Therefore, to better understand the impact of age on gliomagenesis, we asked if adult OPCs can be transformed into gliomas, and if so, what would be the reactivating mechanisms. Using NG2-CreER, a temporally controllable Cre, we introduced p53/NF1 mutations specifically into adult OPCs by giving Tamoxifen (Tmx) to young adult (P45) and aging mice (P180). We found that gliomas formed with high penetrance in both groups. Notably, tumor latency of both groups was very similar (120-180 days post-Tmx), suggesting that OPC transforming potential does not decrease as the brain ages. Adult OPC-derived tumors have pathological features of malignant anaplastic (grade III) glioma and a transcriptional profile similar to human proneural glioma. Interestingly, these features are comparable to gliomas derived from embryonic neural stem cells and perinatal OPCs, indicating that the age at which mutations arise does not influence tumor type. The transcriptional profile of adult OPC-derived tumor cells closely matched that of wild type perinatal OPCs, suggesting that tumor OPCs have been rejuvenated. To identify when mutant OPCs first become reactivated, we analyzed brain regions where these tumors tend to occur (olfactory bulb, thalamus, pyriform cortex, amygdala) at various time points after mutation. At both 50 and 90 days post-Tmx, the number of mutant OPCs was 6-fold higher than that of WT OPCs, indicating pre-malignant over-expansion of these cells. Surprisingly, mutant OPC proliferation was not increased at either time point. Further analysis showed that mutant OPCs have defects in differentiation into mature oligodendrocytes. These results suggest that mutant OPCs become reactivated soon after mutagenesis, and that their inability to differentiate might play a role in malignant transformation. To understand the molecular mechanisms of adult OPC reactivation, we are currently investigating the involvement of signal transduction pathways known to play important roles in early OPC development. In summary, our results show that, despite being mostly quiescent, adult OPCs have the potential to become reactivated by the loss of tumor suppressor genes and can give rise to malignant gliomas. Signaling pathways with known roles in early OPC development might once again play a role in malignant transformation of these cells in the adult brain. Therefore, studying gliomagenesis and OPC development in parallel could lead to novel strategies for treating these deadly tumors. Citation Format: Rui P. Galvao, Roeland G. W. Verhaak, Oded Foreman, Hui Zong. Gliomagenesis via the reactivation of quiescent adult oligodendrocyte precursor cells. [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 351. doi:10.1158/1538-7445.AM2013-351

Abstract 631: Role of thymidylate synthase in genomic instability and tumorigenesis.

Abstract Thymidylate synthase (TS) is a well-established chemotherapy target frequently overexpressed in cancer. Multiple clinical studies demonstrated that high levels of TS correlate with the disease stage and resistance to fluoropyrimidine chemotherapy. Previous studies from our laboratory reassessed the paradigm of TS as a passive proliferation biomarker by providing evidence towards its tumor-promoting capabilities (Cancer Cell 2004; Oncogene 2007). We demonstrated that ectopic TS overexpression combined with loss of tumor suppressor genes significantly enhances the rate and severity of tumorigenesis; however the underlying mechanism of this process remained unclear. TS fuels DNA replication and repair through dTMP and folate biosynthesis. We hypothesized that TS overexpression could become a strong tumor-promoting force by inducing nucleotide imbalance that results in DNA misincorporation and damage. In this study, we analyze effects of TS overexpression in context of aberrant DNA repair and cell cycle misregulation by using Ink4/Arf−/- genetically engineered mouse model. We show that ectopic TS overexpression markedly enhances tumorigenesis in hTS/Ink4/Arf−/- mice compared to Ink4/Arf−/- control resulting in more aggressive tumors. HPLC analyses of mouse tumor-derived cell lines confirmed significant nucleotide imbalance and increase in dTMP, dTDP and dTTP levels in hTS/Ink4/Arf−/- mice compared to control. Analysis of chromosomal changes by spectral karyotyping demonstrated 4 to 5 fold increase in chromosomal aberrations and aneuploidy in hTS positive tumor cell lines. Chromosomal translocations were only found in hTS/Ink4/Arf−/- subtype, indicative of strand-break damage. We assessed extent of damage by Comet assay and yH2AX phosphorylation in mouse tumor cell lines and found increased DNA fragmentation and high yH2AX signal in hTS/Ink4/Arf−/- cells as opposed to Ink4/Arf−/- control. In summary, these studies demonstrate a novel capacity for TS to promote nucleotide imbalance, increase DNA susceptibility to damage and chromosomal aberrations, suggesting a new model where TS overexpression plays a role in the development of genomic instability and tumorigenesis. Citation Format: Lidia V. Kulemina, Min Chen, Kyungah Maeng, Akbar Nawab, Maria Zajac-Kaye. Role of thymidylate synthase in genomic instability and tumorigenesis. [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 631. doi:10.1158/1538-7445.AM2013-631

Abstract 355: Generation of a murine model of Neurofibromatosis Type 2 that accurately recapitulates the spontaneous development of vestibular and spinal schwannomas found in NF2 patients.

Abstract Neurofibromatosis type 2 (NF2) is a genetic disease resulting from germline loss of the NF2 tumor suppressor gene. The hallmark of NF2 is hearing and vestibular loss of function in young adults caused by the development of vestibular schwannomas, which occur bilaterally in nearly all patients and are associated with homozygous loss of NF2. Individuals affected with NF2 also acquire schwannomas of other cranial, spinal and peripheral nerves. Importantly, non-familial schwannomas, the most common tumor of the peripheral nervous system, are also caused by bi-allelic loss of NF2 gene function. A major limitation in the identification of genetic and pharmacologic targets for schwannomas has been the lack of a robust preclinical model that accurately recapitulates human NF2 disease. In order to develop a more robust model, we intercrossed a mouse expressing Cre Recombinase under the control of the 3.9PeriCre promoter with an Nf2 conditional knockout mouse previously generated by Dr. Marco Giovannini to conditionally ablate Nf2 in neural crest-derived cell populations. Histological analysis of the dorsal root ganglion (DRG) and proximal spinal nerves of 3.9PeriCre+;Nf2flox/flox mice revealed that intercrossed mice spontaneously develop tumors between 8 and 10 months at every single spinal level. Microscopic analysis implicated these tumors as schwannomas, as they display a dense pattern of cellularity consistent with the established criteria of GEM schwannoma, including S100β1 positivity. Quantitation of the DRG volume indicated that 3.9PeriCre+ mice have a statistically significant increase when compared to 3.9PeriCre- littermates. FDG-PET studies corroborated this finding, as comparative studies showed an increase in the metabolic activity of paraspinal tumors found in 3.9PeriCre+ mice. Auditory Brainstem Response testing has revealed that 3.9PeriCre+ mice acquire an age-dependent increase in hearing threshold by 8 months when compared to age-matched 3.9PeriCre- controls. Histological analysis of Cranial Nerve VIII confirmed that the hearing loss was directly associated with the development of vestibular schwannomas. To test for concomitant vestibular loss of function, three different behavioral studies (contact righting, trunk curl, and swim) were performed to isolate and test the integrity of the vestibular sense in 3.9PeriCre;Nf2flox/flox mice. These studies indicated that 3.9PeriCre+ mice develop vestibular deficits, as all three studies identified highly significant differences between the two genotypes. Collectively, these genetically engineered mice acquire a physiologic phenotype that closely recapitulates important features of human NF2 disease and provides opportunities for testing putative therapeutic targets using genetic intercrosses or novel small molecule inhibitors. Citation Format: Jeff R. Gehlhausen, Su Jung Park, Matthew Shew, Wade Clapp, Charles Yates. Generation of a murine model of Neurofibromatosis Type 2 that accurately recapitulates the spontaneous development of vestibular and spinal schwannomas found in NF2 patients. [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 355. doi:10.1158/1538-7445.AM2013-355

Neuregulin‐1 overexpression and p53 haploinsufficiency cooperatively promote de novo MPNST pathogenesis

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas arising from neurofibromas in patients with neurofibromatosis type 1 (NF1). We have shown that transgenic mice expressing neuregulin‐1 (NRG1) in Schwann cells (P0‐ GGFβ3 mice) develop neurofibromas and MPNSTs on a C57BL/6 × SJL background. However, on a C57BL/6J background, P0GGFβ3 mice fail to develop tumors despite persistent transgene expression and Schwann cell hyperplasia. Hypothesizing that tumor suppressor gene loss would complement NRG1 overexpression and rescue tumorigenesis, we created cohorts of P0GGFβ3 >;Nf1+/−, P0GGFβ3; Trp53+/− and control (P0‐GGFβ3, Nf1+/−, Trp53+/−) mice and compared their survival and tumorigenesis. The survival of P0GGFβ3; Nf1+/− mice was equivalent to controls and they did not develop tumors. In contrast, P0GGFβ3; Trp53+/− mice died earlier (mean survival, 232 days) and MPNSTs were present in 88.4% of these mice. Further, unlike outbred P0GGFβ3 mice, MPNSTs in P0GGFβ3; Trp53+/− mice arose de novo rather than from neurofibromas. Real‐time PCR, immunohistochemical and sequence analyses showed persistent wild‐type p53 expression. These findings show that NRG1 acts in the same pathways affected by Nf1 loss and suggest that Trp53 haploinsufficiency in the face of NRG1‐driven Schwann cell hyperplasia destabilizes the genome and promotes de novo MPNST tumorigenesis. Funded by NIH and DOD.

Heparin-binding epidermal growth factor-like growth factor eliminates constraints on activated Kras to promote rapid onset of pancreatic neoplasia

Pancreatic cancer remains one of the most deadly cancers with few treatment options at late stages and little information about how it develops through earlier stages. Activating mutation of the Kras gene has been implicated in, but is not sufficient for, tumorigenesis. In mouse models of pancreatic cancer, loss of tumor suppressor genes in conjunction with Kras mutation leads to gradual stochastic acquisition of neoplastic precursors and carcinomas, while many cells remain phenotypically unaltered in younger mice. Here, we demonstrate that two oncogenic events, mutation of Kras and production of the growth factor heparin-binding epidermal growth factor-like growth factor (HB-EGF), are sufficient for rapid and complete neoplastic transformation of the exocrine pancreas. We found that macrophages are the major source of HB-EGF production in pancreatic cancer tissue samples and that macrophages are present in high density and in close association with human pancreatic cancer lesions. In a mouse model, high macrophage density was observed at the earliest stages of neoplastic transformation. The consequence of elevated HB-EGF signaling was investigated without the confounding effects of other macrophage-produced factors via transgenic overexpression of the active form of HB-EGF. In this model, HB-EGF was sufficient to promote Kras-initiated tumorigenesis, inducing rapid and complete neoplastic transformation of the entire exocrine pancreas shortly after birth. HB-EGF overexpression and KrasG12D together, but neither alone, increased proliferation with increased CyclinD1 and decreased Cdkn2a/2d (p16/p19Ink4A/Arf). These findings establish the importance of oncogenic synergy in cancer initiation and promotion and establish a molecular link between inflammation and the earliest stages of tumor induction.

Abstract IA5: Genetics of clonal progression in pancreatic cancer

Abstract The goal of our laboratory is to understand the extent to which genetic features of a neoplasm influence its metastatic progression. Towards this goal we have focused on human pancreatic ductal adenocarcinoma (PDAC) as it represents an ideal tumor type for study of the metastatic process. For example, the precursor lesions that give rise to PDAC have been described in detail (pancreatic intraepithelial neoplasia, or PanIN), exomic sequencing has elucidated the major genetic alterations of this tumor type, and the accumulation of these genetic alterations during PanIN formation underlies the well accepted genetic progression model of pancreatic ductal carcinogenesis. Finally, PDAC is a tumor type that is often diagnosed in the advanced stages of disease and thus most patients have their primary carcinoma in situ for comparison to matched metastatic disease. Such is not the case for many other common human tumor types. We have taken the unique approach of performing rapid autopsies for acquisition of primary and metastatic cancer tissues from a large number of individuals, and have used this resource to make several novel observations related to the timing of acquisition of metastatic traits. First, not all pancreatic cancers are metastatic; of those that do form metastases the range of deposits seen at autopsy ranges from few (<10, oligometastatic disease) to >100 (widely metastatic). Moreover, loss of the SMAD4 tumor suppressor gene that occurs during carcinogenesis is highly correlated with widespread metastatic disease. Second, using a whole exome sequencing approach we have defined the clonal evolution of PDAC. This has shown that the majority of genetic alterations are founder mutations in that they are encompassed with the parental clone of cells that give rise to PDAC, upon which are superimposed additional genetic alterations that accumulate during subclonal evolution. Geographic mapping of subclones based on their unique genetic signatures indicates that metastatic subclones originate within the primary carcinoma; moreover, several years are required for development of metastatic subclones beyond initiation of the infiltrating carcinoma by the parental clone. Thus, even in carcinomas with SMAD4 loss additional events are needed for successful metastasis to occur. Most recently, we have investigated pancreatic cancer progression by utilizing a mathematical framework of metastasis formation together with comprehensive data of PDAC patients obtained at autopsy. This work indicates that primary and metastatic PDAC growth is initially exponential, and that patients with seemingly early stage disease likely harbor metastases at diagnosis. Furthermore, based on the estimating rates of PDAC growth and dissemination, we analyzed the effects of different treatment modalities and found that therapies which efficiently reduce the growth rate of cells earlier in the course of treatment appear to be superior to upfront tumor resection. These lines of investigation not only shed light on this difficult tumor type but also have implications for most other major tumor types for which metastatic progression is common. Citation Format: Christine A. Iacobuzio-Donahue. Genetics of clonal progression in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr IA5.

The prognostic significance and value of cyclin D1, CDK4 and p16 in human breast cancer

IntroductionLoss of the retinoblastoma protein tumor suppressor gene (RB) coding for a nuclear phosphoprotein that regulates the cell cycle is found in many human cancers and probably leads to disruption of the p16-cyclin D1-CDK4/6-RB pathway. Cyclin D1 is known to activate CDK4, which then phosphorylates the RB protein, leading to cell cycle progression. p16 inhibits CDK4, keeping RB hypophosphorylated and preventing cell cycle progression. The significance of these three markers, cyclin D1, CDK4 and p16, for breast cancer and carcinogenesis is nevertheless still controversial.MethodsThe material consisted of 102 formalin-fixed human breast cancer samples, in which cyclin D1, CDK4 and p16 expression was evaluated immunohistochemically. The amounts of cyclin D1 mRNA present were analyzed by quantitative real time PCR.ResultsHigh cyclin D1 expression statistically significantly correlated with lower tumor grade, estrogen and progesterone receptor positivity and lower proliferation activity in breast tumors and increased breast cancer-specific survival and overall survival. Tumors with high cyclin D1 protein had 1.8 times higher expression of cyclin D1 mRNA. CDK4 expression did not correlate with cyclin D1 expression or the survival data. p16 expression was associated with Human Epidermal Growth Factor Receptor 2 (HER2) negativity and increased breast cancer-specific survival and disease-free survival. No statistical correlations between cyclin D1, CDK4 and p16 were found.ConclusionsCyclin D1 was associated with a good breast cancer prognosis but functioned independently of CDK4. High cyclin D1 expression may be partially due to increased CCND1 transcription. p16 correlated with a better prognosis and may function without CDK4. In conclusion, it appears that cyclin D1, CDK4 and p16 function independently in human breast cancer.

Burkitt Lymphoma Arising From Lymphoplasmacytic Lymphoma Following Acquisition of MYC Translocation and Loss of the ETV6 Tumor Suppressor Gene

Lymphoplasmacytic lymphoma is a mature B-cell lymphoma with variable plasmacytic differentiation that displays an indolent clinical course. Its transformation to a high-grade B-cell lymphoma may occur uncommonly. Although acquisition of a MYC translocation could result in transformation of a low-grade lymphoma into diffuse large B-cell lymphoma, Burkitt lymphoma, or B-lymphoblastic leukemia, to our knowledge the latter 2 transformations have not been well documented in lymphoplasmacytic lymphoma. We report the case of a 70-year-old woman with a 9-year history of lymphoplasmacytic lymphoma/Waldenström macroglobulinemia who presented with rapid enlargement of a left neck mass and pancytopenia, which was diagnosed as Burkitt lymphoma with extensive bone marrow involvement. A series of histopathologic, molecular, and cytogenetic evaluations proved a cytogenetic evolution including t(8;14)(q24;q32)/MYC-IgH and identical clonal B-cell gene rearrangements from the 2 distinct lymphomas, confirming stage 4 aggressive Burkitt lymphoma arising from lymphoplasmacytic lymphoma.

Identification and Pattern Analysis of SNPs Involved in Colorectal Cancer

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths globally posing a lifetime risk of 80-100% in every individual. Genetics and relevant mechanisms underlying some key signaling pathways like Wnt, TGF, p53, K-ras etc. play a detrimental role in governing the predisposition for CRC. A high percentage of colorectal tumors (adenomas and carcinomas) show activating mutations in beta-catenin or axin, whereas, loss of certain tumor suppressor genes (TSGs), like APC cause the initiation of random polyps in the colon. All of these molecules incidentally are critical components of an evolutionarily conserved Wnt signaling pathway, which is instrumental at various time-points in the development of this disease. Differences in SNP profiles amongst sample groups in the genomic landscape can be recognized through a smart and efficient use of machine learning techniques. The statistics and pattern analyses of these SNP profiles, interestingly provides us with a concrete and logical platform upon which, relative contribution/s of each unique SNP, ranging “from cause to effect” can be significantly assessed. The biological relevance of these SNP variations with respect to cancer prediction and predisposition, however, remains to be resolved, pending a better understanding of the impact of rational control design in SNP studies. Our results emerging from the analyses of significant SNPs reported here, demonstrates the utility of relevant bioinformatics tools and machine learning techniques in discriminating diseased populations based on realistic SNP data. In this study, we have primarily targeted critical members of Wnt signaling pathway, which play important developmental role/s during different stages of colorectal cancer, depicting a classical “multigene-multistep nature” of cancer. We have identified and related common genetic variants for the “early-acting” and “late-acting” members of this pathway, that are most prevalent in patients with CRC disease, by harnessing the power of developmental biology tools. In addition, complex relationships and correlations hidden in large data-sets have been dug and analyzed here, by deploying various datamining (bioinformatics) techniques. The report discusses the scope of such a combinatorial approach, by identifying some potential candidate targets of therapy, in translational research and clinical medicine interventions.

Proteostasis Modulators Prolong Missense VHL Protein Activity and Halt Tumor Progression

Although missense mutations of the von Hippel-Lindau disease (VHL) gene are the most common germline mutation underlying this heritable cancer syndrome, the mechanism of tumorigenesis is unknown. We found a quantitative reduction of missense mutant VHL protein (pVHL) in tumors associated with physiologic mRNA expression. Although mutant pVHL is unstable and degraded contemporarily with translation, it retains its E3 ligase function, including hypoxia-inducible factor degradation. The premature pVHL degradation is due to misfolding and imbalance of chaperonin binding. Histone deacetylase inhibitors (HDACIs) can modulate this pathway by inhibiting the HDAC-Hsp90 chaperone axis, stabilizing pVHL, and restoring activity comparable to wild-type protein, both in vitro and in animal models. Furthermore, HDACI-mediated stabilization of missense pVHL significantly attenuates the growth of 786-O rodent tumor model. These findings provide direct biological insight into VHL-associated tumors and elucidate a treatment paradigm for VHL.