Homozygous Deletion Of P53 Research Articles

Abstract C37: Setdb1 is required for formation of pancreatic ductal adenocarcinoma by inhibiting apoptosis through regulation of p53 expression

Abstract Epigenetics is one of the representative mutation signatures in human pancreatic ductal adenocarcinoma (PDAC). Histone modification, one group of epigenetic regulators, is known to play an important role in cancer development. Setdb1, a histone methyltransferase that trimethylates histone H3 on lysine 9, has been reported to promote tumorigenesis by altering the expression of oncogenes/ tumor suppressor genes. In this study, we aimed to elucidate the functional role of Setdb1 in pancreatic acinar regeneration and PDAC formation. Setdb1 was expressed in pancreatic ductal cells and a subset of pancreatic acinar cells in wild-type mice. In pancreatic specific Setdb1 conditional KO mice, no abnormality was observed in pancreatic development. However, pancreatic deletion of Setdb1 resulted in pancreatic atrophy concomitant with increased apoptosis and upregulated p53 expression after cerulein-induced pancreatitis. To investigate the impact of pancreatic Setdb1 deletion in the context of oncogenic Kras, we next generated Ptf1a-Cre; KrasG12D; Setdb1f/f (KCS) mice and compared to control Ptf1a-Cre; KrasG12D (KC) mice. Pancreatic deletion of Setdb1 significantly accelerated spontaneous development of acinar-to-ductal metaplasia (ADM) and pancreatic intraepitherial neoplasia (PanIN) in KCS mice at the age of 4 weeks. Additionally, immunostaining revealed a dramatic increase in apoptotic cells and upregulated p53 expression in KCS mice. Consistently, microarray analysis revealed upregulated expression of apoptotic signaling pathway and p53 downstream gene pathway in KCS mice compared to KC mice. Furthermore, Chromatin immunoprecipitation experiments in acinar cells isolated from wild-type mice revealed that Setdb1 bound to the promoter regions of p53 to support its expression. Given that Setdb1 loss caused increased apoptosis mediated by p53, we next examined the impact of pancreatic Setdb1 deletion on PDAC formation in the context of heterozygous p53 deletion. Surprisingly, PDAC formation was suppressed in KCS mice in the context of heterozygous p53 deletion (KPheteroCS mice). Again, apoptosis was increased and p53 expression was upregulated in KPheteroCS mice compared to KPheteroC mice. Furthermore, KPheteroCS mice showed significantly better prognosis for survival compared to control KPheteroC mice. In sharp contrast, in KCS mice in the context of homozygous p53 deletion, PDAC was formed and increased apoptosis was cancelled. Additionally, suppression of Setdb1 in human p53 wild PDAC cells led to p53-induced apoptosis. These findings suggested that Setdb1 deletion protects against PDAC formation with inducing apoptosis through regulation of p53 expression. In conclusion, Setdb1 is required for normal pancreatic acinar regeneration and is required for PDAC formation by inhibiting apoptosis through regulation of p53 expression. Thus, Setdb1 could be a therapeutic target for PDAC that retains p53 function. Citation Format: Satoshi Ogawa, Akihisa Fukuda, Makoto Sono, Yuichi Fukunaga, Tomonori Masuda, Osamu Araki, Munemasa Nagao, Takaaki Yoshikawa, Yukiko Hiramatsu, Motoyuki Tsuda, Takahisa Maruno, Yuki Nakanishi, Hiroshi Seno. Setdb1 is required for formation of pancreatic ductal adenocarcinoma by inhibiting apoptosis through regulation of p53 expression [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C37.

Abstract 951: Setdb1 deletion protects against formation of pancreatic ductal adenocarcinoma through inducing apoptosis and upregulating p53 expression

Abstract One group of epigenetic regulators known to be altered in cancer development is histone modification. Setdb1, a histone methyltransferase that trimethylates histone H3 on lysine 9 (H3K9), has been implicated in tumorigenesis by altering the expression of oncogenes/ tumor suppressor genes. In this study, we aimed to elucidate the functional role of Setdb1 in pancreatic acinar regeneration and formation of pancreatic ductal adenocarcinoma (PDAC). Setdb1 was expressed in pancreatic ductal cells and a small subset of pancreatic acinar cells in adult wild type mice. Upon caerulein-induced pancreatitis, Setdb1 was expressed in acinar cells and acinar to ductal metaplasia (ADM). To determine the impact of pancreatic Setdb1 deletion on acinar regeneration, we generated Ptf1a-Cre; Setdb1f/f (CS) mice and induced caerulein-pancreatitis. Pancreatic deletion of Setdb1 resulted in pancreatic atrophy concomitant with increased apoptosis of acinar cells and increased expression of p53 in CS mice after caerulein-induced pancreatitis. In addition, pancreatic acinar regeneration was delayed with persistent inflammation inCS mice. To investigate the impact of pancreatic Setdb1 deletion on PDAC formation, we next generated Ptf1a-Cre; KrasG12D; Setdb1f/f (KCS) mice and compared to control Ptf1a-Cre; KrasG12D (KC) mice. Pancreatic deletion of Setdb1 dramatically accelerated spontaneous development of ADM and pancreatic intraepithelial neoplasia (PanIN), the most common precursor lesion of PDAC, in KCS mice compared to KC mice at the age of 4 weeks. Acinar cell culture experiments revealed that Setdb1 null acinar cells converted to ADM, at least in part, in a cell autonomous manner. Long term observation revealed massive pancreatic atrophy in KCS mice. Immunostaining revealed a dramatic increase in apoptotic cells and upregulated expression of p53 in KCS mice compared to KC mice at the age of 20 weeks. Consistently, microarray analysis revealed upregulated expression of p53 and apoptotic pathway genes in KCS mice compared to KC mice. We next examined the impact of pancreatic Setdb1 deletion on PDAC formation in the context of p53 deletion. Surprisingly, PDAC formation was suppressed in KCS mice in the context of heterozygous p53 deletion (KPhereroCS mice). Again, apoptosis was increased and p53 expression was upregulated in KPhereroCS mice. In contrast, in the context of homozygous p53 deletion, PDAC was formed and increased apoptosis was canceled in KPhomoCS mice. These findings suggested that Setdb1 deletion protects against PDAC formation through inducing apoptosis, which is mediated by p53. In conclusion, Setdb1 is required for normal pancreatic acinar regeneration and protects against PDAC formation though inducing apoptosis, which is mediated by p53. Thus, Setdb1 could be a therapeutic target for PDAC which retains wild p53 expression. Citation Format: Satoshi Ogawa, Akihisa Fukuda, Motoyuki Tsuda, Tomonori Masuda, Makoto Sono, Yuichi Fukunaga, Takaaki Yoshikawa, Osamu Araki, Munemasa Nagao, Norihiro Goto, Yukiko Hiramatsu, Takahisa Maruno, Yoichi Shinkai, Hiroshi Seno. Setdb1 deletion protects against formation of pancreatic ductal adenocarcinoma through inducing apoptosis and upregulating p53 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 951.

Abstract 4147: DCLK1 a novel therapeutic target in non-small cell lung cancer

Abstract Introduction: Lung cancers possess a rare population of cells that have the capacity to self-renew, differentiation and ultimately promote tumorigenesis. These cells are often resistance to conventional therapies and are responsible for tumor relapse and metastatic spread, which contribute to the overall mortality observed in non-small cell lung cancer (NSCLC). These cells are termed tumor stem cells (TSCs) and as such are highly desirable targets for novel therapeutic strategies. Doublecortin-like Kinase 1 (DCLK1) has recently been identified as a tumor stem cell marker in the intestine and pancreas and is dysregulated in many solid tumor cancers including lung cancer. Increased expression has been demonstrated to correlate with poor survival in several solid tumor cancers. Thus these data taken together provide the rational for targeting DCLK1 expressing cells in NSCLC. Methods: Small interfering RNA (siRNA) against DCLK1 was transfected into NSCLC cell lines (A549 - wt-p53; H1299 –homozygous deletion of p53) of highly drug resistant (A549) and metastatic (H1299). Cell proliferation, colony formation, self-renewal, cell migration and invasion and drug resistance assays were performed to assess the tumor stem cell characteristics. A549 cells were treated with siDCLK1 and cisplatin in order to assess the role of DCLK1 to reverse the drug-resistance. A549 cells were utilized to generate tumor xenograft in order to assess the role of DCLK1 knockdown on NSCLC tumorigenesis in vivo. The data were generated utilizing experimental protocols for clonogenic culture, immunohistochemistry, RT-qPCR and Western blotting. Results: Silencing DCLK1 via RNA interference decreased the colony formation (p<0.001), and self-renewal ability (p<0.0001) of the NSCLC cell lines in vitro. Knocking down DCLK1 reduced (p<0.001) NSCLC cells migration and invasion in vitro. Furthermore, DCLK1 knockdown decreased the expression of EMT associated factors (Slug, Snail, Twist, Zeb1/2, Vimentin) and pluripotency factors. Initial cisplatin treatment results in the isolation of cisplatin resistant A549 cells. Retreatment of cisplatin resistant A549 cells results in greater resistance and survival ability. However, combination therapy of these resistance cells with siDCLK1 and cisplatin overcame cisplatin-induced resistance, resulting in greater cell death compared to cisplatin or siDCLK1 alone treatments. Nanoparticle encapsulated siDCLK1 treatment in xenograft tumors resulted in tumor growth arrest in vivo. Conclusions: NSCLC cells highly express DCLK1 display enhanced self-renewal, increased migration and invasion. Combination therapy of siDCLKL1 with cisplatin overcame cisplatin induced drug resistance. Targeting DCLK1 reduced tumor xenograft in vivo. Taken together these data suggest that DCLK1 inhibition reduces TSC related properties, overcome drug resistance and inhibits tumor growth, thus making DCLK1 targeted therapy an attractive tool for combating NSCLC. Citation Format: Parthasarathy Chandrakesan, Dongfeng Qu, Randal May, Nathaniel Weygant, William Berry, Naushad Ali, Sripathi Sureban, Eddie Bannerman-Menson, Michael Bronze, Courtney Houchen. DCLK1 a novel therapeutic target in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4147. doi:10.1158/1538-7445.AM2017-4147

Abstract 1845: Mutant p53 promotes progression and metastasis of mouse oral tumors induced by 4NQO, associated with specific immune infiltrates

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. The vast majority of the human HNSCCs contain p53 mutations, some of which acquire oncogenic gain-of-function (GOF) activities. Previous mouse models in which either the p53R172H GOF mutation or deletion of the p53 gene were co-activated with oncogenic K-ras showed that p53R172H can predispose to oral tumor initiation, accelerate tumor growth and promote progression to SCC. However, the impact of p53 gain- and loss-of-function (LOF) mutations in metastasis could not be assessed because of the low rates of progression to SCC in that model. To overcome this limitation, we used the tobacco-surrogate 4NQO to induce oral lesions that may progress to advance carcinomas following stepwise changes that resemble the gradual accumulation of histological and molecular abnormalities observed during human oral cancer progression. To examine the role of p53 mutations during SCC malignant progression we applied 4NQO to mice in which the p53R172H mutation or homozygous deletion of p53 were activated in oral epithelial cells. We observed that oral tumors appeared faster in the presence of p53 mutations than in mice with wtp53. No significant difference was observed between the p53R172H mutation and p53 deletion. However, survival was shorter in mice expressing p53R172H than in mice with loss of p53. Notably, regional and distant metastasis were only observed in mice with oral tumors expressing p53R172H. Therefore, these mice are excellent tools for pre-clinical studies designed to target early stages of tumor development or latest stages of progression and metastasis. As immunotherapy is becoming adopted as part of the standard treatment for head and neck cancer patients, we conducted a detailed characterization of the immune infiltrates of the lesions that developed in these mice in order to assess their immunogenicity. We observed a progressive increase in T-cell infiltration from normal oral mucosa to oral premalignant lesions (OPL) to SCC, in the presence or absence of p53 mutations. Similarly, the expression of immune checkpoint proteins, including PD1, PDL1 and CTLA4 increased during oral tumor progression, regardless of the p53 status. Interestingly, we observed a sharp increase in the presence of immune infiltrates expressing the co-stimulatory OX40 in SCCs relative to premalignant lesions, with different extent depending on the nature of the p53 mutation. The significance of these observations is currently being analyzed. In summary, p53R172H promotes metastasis in oral tumors induced by 4NQO. In addition, T-cell infiltration and activation of immune checkpoints during oral tumor progression suggest that this is an excellent model to test immunotherapy-based strategies. Furthermore, our findings suggest that OX40-based therapies may be tailored to patients according to their p53 status. Citation Format: Jin Wang, Yuanyuan Zhang, Bingbing Wang, Adel K. El-Naggar, Jeffrey N. Myers, Carlos Caulin. Mutant p53 promotes progression and metastasis of mouse oral tumors induced by 4NQO, associated with specific immune infiltrates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1845. doi:10.1158/1538-7445.AM2017-1845

P53 Activity Results in DNA Replication Fork Processivity

p53 induces cell death upon DNA damage, but this may not confer all of its tumor suppressor activity. We report that p53 activation enhances the processivity of DNA replication, as monitored by multi-label fiber assays, whereas removal of p53 reduces fork progression. This is observed in tumor-derived U2OS cells but also in murine embryonic fibroblasts with heterozygous or homozygous p53 deletion and in freshly isolated thymocytes from mice with differential p53 status. Mdm2, a p53-inducible gene product, similarly supports DNA replication even in p53-deficient cells, suggesting that sustained Mdm2-expression is at least one of the mechanisms allowing p53 to prevent replicative stress. Thus, p53 helps to protect the genome during S phase, by preventing the occurrence of stalled or collapsed replication forks. These results expand p53's tumor-suppressive functions, adding to the ex-post model (elimination of damaged cells) an ex-ante activity; i.e., the prevention of DNA damage during replication.

Bone morphogenetic protein signaling promotes tumorigenesis in a murine model of high-grade glioma.

Improved therapies for high-grade glioma (HGG) are urgently needed as the median survival for grade IV gliomas is only 15 months. Bone morphogenetic protein (BMP) signaling plays critical and complex roles in many types of cancer, including glioma, with most of the recently published work focusing on BMP-mediated regulation of glioma stem cells (GSCs). We hypothesized that BMP signaling may be an important modulator of tumorigenic properties in glioma cells outside of the GSC compartment. We used a human HGG tissue microarray and performed immunohistochemistry for phospho-Smads1,5,8. To examine the role of BMP signaling in tumorigenic astrocytes, transgenic mice were used to delete the BMP type IA receptor (Bmpr1a) and generate astrocytes transformed with oncogenic Ras and homozygous deletion of p53. The cells were transplanted orthotopically into immunocompetent adult host mice. First we established that BMP signaling is active within the vast majority of HGG tumor cells. Mice implanted with BMPR1a-knockout transformed astrocytes showed an increase in median survival compared with mice that received BMPR1a-intact transformed astrocytes (52.5 vs 16 days). In vitro analysis showed that deletion of BMPR1a in oncogenic astrocytes resulted in decreased proliferation, decreased invasion, decreased migration, and increased expression of stemness markers. In addition, inhibition of BMP signaling in murine cells and astrocytoma cells with a small molecule BMP receptor kinase inhibitor resulted in similar tumor suppressive effects in vitro. BMP inhibition may represent a viable therapeutic approach in adult HGG.

Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model.

Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSA sT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult Ubc CreERT2; ROSA sT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult Ubc CreERT2; ROSA sT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1 CreERT2/+; ROSA sT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1 CreERT2/+; ROSA sT and Atoh1 CreERT2/+; ROSA sT; p53f lox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting.

Igf2 pathway dependency of the Trp53 developmental and tumour phenotypes.

Insulin-like growth factor 2 (IGF2) and the transformation related protein 53 (Trp53) are potent regulators of cell growth and metabolism in development and cancer. In vitro evidence suggests several mechanistic pathway interactions. Here, we tested whether loss of function of p53 leads to IGF2 ligand pathway dependency in vivo. Developmental lethality occurred in p53 homozygote null mice that lacked the paternal expressed allele of imprinted Igf2. Further lethality due to post-natal lung haemorrhage occurred in female progeny with Igf2 paternal null allele only if derived from double heterozygote null fathers, and was associated with a specific gene expression signature. Conditional deletion of Igf2fl/fl attenuated the rapid tumour onset promoted by homozygous deletion of p53fl/fl. Accelerated carcinoma and sarcoma tumour formation in p53+/− females with bi-allelic Igf2 expression was associated with reductions in p53 loss of heterozygosity and apoptosis. Igf2 genetic dependency of the p53 null phenotype during development and tumour formation suggests that targeting the IGF2 pathway may be useful in the prevention and treatment of human tumours with a disrupted Trp53 pathway.

KrasG12D and p53 Mutation Cause Primary Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma (IHCC) is a primary cancer of the liver with an increasing incidence and poor prognosis. Preclinical studies of the etiology and treatment of this disease are hampered by the relatively small number of available IHCC cell lines or genetically faithful animal models. Here we report the development of a genetically engineered mouse model of IHCC that incorporates two of the most common mutations in human IHCC, activating mutations of Kras (Kras(G12D)) and deletion of p53. Tissue-specific activation of Kras(G12D) alone resulted in the development of invasive IHCC with low penetrance and long latency. Latency was shortened by combining Kras(G12D) activation with heterozygous or homozygous deletion of p53 (mean survival of 56 weeks vs. 19 weeks, respectively), which also resulted in widespread local and distant metastasis. Serial analysis showed that the murine models closely recapitulated the multistage histopathologic progression of the human disease, including the development of stroma-rich tumors and the premalignant biliary lesions, intraductal papillary biliary neoplasms (IPBN), and Von Meyenburg complexes (VMC; also known as biliary hamartomas). These findings establish a new genetically and histopathologically faithful model of IHCC and lend experimental support to the hypothesis that IPBN and VMC are precursors to invasive cancers.

A Novel Transgenic Animal Model for HPV‐Associated Cancer

Objective: The incidence of HPV-associated tumors is rising at a rapid pace. In order to develop novel therapies with high efficacy but low toxicity, it is crucial to have an animal model in which HPV-associated oral lesions spontaneously develop. Our objective was to develop such a model. Method: A transgene was created to target expression of E7 to the oral cavity by cloning HPV-16 E7 downstream of the L2 promoter. Transgenic mice which carry this gene were created on the C57Bl6 background. Then, these mice were backcrossed with a strain of mice heterozygous for p53. Results: Mice were bred to create the desired genotype, E7 +/+, p53 -/-. Because of the short life span and limited breeding potential of mice with a homozygous p53 deletion, breeding was performed with p53 heterozygous mice. In accordance with Mendelian genetics, we found the majority of offspring were heterozygous for p53, and did not form spontaneous tumors. Of the offspring with homozygous p53 deletions, few lived longer than 25 weeks. However, one mouse lived long enough to develop an HPV-associated oral tumor. E7 transcript was detected in the tumor by RT-PCR, and E7 protein was detected via Western blot. Conclusion: We report the development of a transgenic mouse which spontaneously expressed an HPV-associated oral tumor. Additional genetic manipulation may be required to enhance the longevity and stability of this mouse strain. This strain has the potential to serve as a platform to investigate novel therapeutic strategies for HPV-associated tumors.

Genetic instability and mammary tumor formation in mice carrying mammary-specific disruption of Chk1 and p53

Checkpoint kinase 1 (Chk1) is a key element in the DNA-damage response pathway that is required for maintaining genomic stability. To study the potential role of Chk1 in mammary tumorigenesis, we disrupted it using a Cre/loxP system. We showed that although Chk1 heterozygosity caused abnormal development of the mammary gland, it was not sufficient to induce tumorigenesis. Simultaneous deletion of one copy of p53 failed to rescue the developmental defects; however, it synergistically induced mammary tumor formation in Chk1(+/-);MMTV-Cre animals with a median time to tumor latency of about 10 months. Chk1 deficiency caused a preponderance of abnormalities, including prolongation, multipolarity, misalignment, mitotic catastrophe and loss of spindle checkpoint, that are accompanied by reduced expression of several cell cycle regulators, including Mad2. On the other hand, we also showed that Chk1 deficiency inhibited mammary tumor formation in mice carrying a homozygous deletion of p53, uncovering a complex relationship between Chk1 and p53. Furthermore, inhibition of Chk1 with a specific inhibitor, SB-218078, or acute deletion of Chk1 using small hairpin RNA killed mammary tumor cells effectively. These data show that Chk1 is critical for maintaining genome integrity and serves as a double-edged sword for cancer: although its inhibition kills cancer cells, it also triggers tumorigenesis when favorable mutations are accumulated for cell growth.

Abstract 1964: Genetic instability and mammary tumor formation in mice carrying mammary-specific disruption of Chk1

Abstract Chk1 is a key element in the DNA damage response pathway that is required for maintaining genomic stability. To study the potential role of Chk1 in mammary tumorigenesis, we disrupted it using a Cre/loxP system. We showed that although Chk1 haploinsufficiency caused abnormal development of the mammary gland, it was not sufficient to induce tumorigenesis. Simultaneous deletion of one copy of p53 failed to rescue the developmental defects; however, it synergistically induced mammary tumor formation in Chk1+/-;MMTV-Cre animals with a median time to tumor latency of about 10 months. Chk1 deficiency caused a preponderance of abnormalities, including prolongation, multipolarity, misalignment, mitotic catastrophe and loss of spindle checkpoint that is accompanied by reduced expression of several cell cycle regulators, including Mad2. On the other hand, we also showed that Chk1 deficiency inhibited mammary tumor formation in mice carrying a homozygous deletion of p53, uncovering a complex relationship between Chk1 and p53. Furthermore, inhibition of Chk1 with a specific inhibitor, SB-218078, or acute deletion of Chk1 using shRNA killed mammary tumor cells effectively. These data demonstrate that Chk1 is critical for maintaining genome integrity and serves as a double-edged sword for cancer: while its inhibition kills cancer cells, it also triggers tumorigenesis when avorable mutations are accumulated for cell growth. 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 1964.

The Cell Line Secretome, a Suitable Tool for Investigating Proteins Released in Vivo by Tumors: Application to the Study of p53-Modulated Proteins Secreted in Lung Cancer Cells

Malignant processes such as metastasis, invasion, or angiogenesis are tightly dependent on the composition of the extracellular medium, which is itself affected by the release of proteins by the tumor cells. p53, a major tumor suppressor protein very frequently mutated and/or inactivated in cancer cells, is known to modulate the release of proteins by the tumor cells; however, while p53-modulated intracellular proteins have been extensively studied, little is known concerning their extracellular counterparts. Here, we characterized the p53-dependent secretome of a lung tumor model in vitro (H358 human nonsmall cell lung adenocarcinoma cell line with a homozygous deletion of p53) and demonstrate that the modulation of exported proteins can also be detected in vivo in the plasma of tumor-bearing mice. We used a clone of H358, stably transfected with a tetracycline-inducible wild-type p53-expressing vector. With the use of iTRAQ labeling and LC-MALDI-MS/MS analysis, we identified 909 proteins released in vitro by the cells, among which 91 are p53-modulated. Three proteins (GDF-15, FGF-19, and VEGF) were also investigated in H358/TetOn/p53 xenograft mice. The ELISA dosage on total tumor protein extracts confirmed the influence of p53 on the release of these proteins in vivo. Moreover, the GDF-15 concentration was measured in the plasma and its p53-dependent modulation was confirmed. To our knowledge, this is the first report establishing that the in vitro cell line secretome is reliable and reflects the extracellular release of proteins from tumor cells in vivo and could be used to identify putative tumor markers.

Targeted mutation of p53 and Rb in mesenchymal cells of the limb bud produces sarcomas in mice

Mice bearing germ line mutations of p53 develop sarcomas at a significant rate. Since they are susceptible to a variety of other malignancies, they are not ideally suited to the study of sarcomas. To test the possibility that targeted mutation of tumor suppressor genes in early mesenchymal cells would induce formation of sarcomas, the Prx1-cre transgenic mouse was crossed to mice-bearing floxed alleles of p53 and Rb. Mice with homozygous deletion of p53 (Prx1-cre p53(lox/lox)) developed sarcomas in the extremities at a mean time of 50 weeks. Osteosarcomas (OS) were the most common type of sarcoma (61%) followed by poorly differentiated soft tissue sarcomas (PDSTS) (32%). Homozygous deletion of p53 produced sarcomas significantly more rapidly than heterozygous deletion, which resulted in sarcoma formation after a mean of 96 weeks. Mice with homozygous Rb mutation (Prx1-cre Rb(lox/lox)) developed normally and had no ostensible defects in the limbs. In contrast to p53, targeted deletion of Rb did not produce sarcomas in the limbs. However, simultaneous deletion of Rb and p53 accelerated the time to sarcoma formation, and a greater percentage of PDSTS were found. Deletion of p53 in committed osteoblasts by the Col1a1-cre transgenic mouse bearing an osteoblast-specific enhancer resulted in a high percentage of OS. These findings suggest that deletion of p53 in mesenchymal cells that give rise to osteoblasts is a powerful initiator of OS. Deletion of Rb does not initiate sarcoma formation in mice, but it accelerates formation of both soft tissue sarcomas and OS.

The role of p53 loss in human cancer progression and therapeutic response

Understanding the roles somatic mutations play in cancer progression is a critical step towards the development of novel treatment strategies. The p53 tumor suppressor gene is the most commonly mutated gene in human cancer. A non‐tumorigenic, human cell line model system to study p53 loss in the absence of other oncogenic changes has not been available. Such a model system is key to investigating the possible direct causal connection that p53 deficiency may have in human cancers and to discovering the potential therapeutic effects of such loss. To evaluate the relevance of p53 loss, we have generated a novel in vitro model system using somatic cell knock‐out technology. This knock‐out technology, involving targeted homologous recombination, has enabled us to delete p53 in the non‐tumorigenic epithelial cell line, MCF10A. The initial hypothesis to be tested is that loss of p53 will cause a non‐tumorigenic cell line to display a more oncogenic phenotype. Recently, the homozygous deletion of p53 in four MCF10A clones was confirmed. While MCF10A cells have the appropriate G1/S and G2/M checkpoint responses upon DNA damage, the p53‐/‐ clones display a defective G1/S phase checkpoint response. Further characterization of each clone is being performed to assess the utility of p53 loss as a potential biomarker to predict the therapeutic response of cells treated with current chemotherapeutic regimens.

PNC‐28, a p53‐derived peptide that is cytotoxic to cancer cells, blocks pancreatic cancer cell growth in vivo

PNC-28 is a p53 peptide from its mdm-2-binding domain (residues 17-26), which contains the penetratin sequence enabling cell penetration on its carboxyl terminal end. We have found that this peptide induces necrosis, but not apoptosis, of a variety of human tumor cell lines, including several with homozygous deletion of p53, and a ras-transformed rat acinar pancreatic carcinoma cell line, BMRPA1. Tuc3. On the other hand, PNC-28 has no effect on untransformed cells, such as rat pancreatic acinar cells, BMRPA1, and human breast epithelial cells and no effect on the differentiation of human stem cells. In this study, we now test PNC-28 in vivo for its ability to block the growth of BMRPA1. Tuc3 cells. When administered over a 2-week period in the peritoneal cavities of nude mice containing simultaneously transplanted tumors, PNC-28 causes complete destruction of these tumors. When delivered concurrently with tumor explantation at a remote site, PNC-28 causes a complete blockade of any tumor growth during its 2-week period of administration and 2 weeks posttreatment, followed by weak tumor growth that plateaus at low tumor sizes compared with tumor growth in the presence of a control peptide. When administered after tumor growth has occurred at a site remote from the tumor, PNC-28 causes a decrease in tumor size followed by a slow increase in tumor growth that is significantly slower than growth in the presence of control peptide. These results suggest that PNC-28 may be effective in treating cancers especially if delivered directly to the tumor.

Clinicopathologic markers of uterine leiomyosarcoma originating from smooth muscle tumors of low malignancy

The aims of the study were (1) to increase the limited knowledge on molecular markers contributing to uterine leiomyosarcoma (LMS) tumorigenesis and (2) to test the ability to predict the clinical course of smooth muscle tumors of low malignancy (SMTLM). Retrospectively, 2,360 uterine smooth muscle tumors were classified according to the Stanford criteria. After central review, the clinical course of the SMTLM was traced, and immunohistochemical stainings for p53, p16, p21, Rb, estrogen receptor, progesterone receptor (PR), cyclin A, cyclin D1, vascular endothelial growth factor, WT-1, and Ki-67 were performed on paraffin-embedded tissue of the primary tumor and recurrences. On the same tissues, fluorescence in situ hybridization (FISH) analysis was performed to determine differences in HMGA2, p53, Rb, and 1p36 loci involvement. Five SMTLM were identified, of which two relapsed after 2 and 3 years, respectively. Recurrence tumors were surgically resected, and 3 years later, both patients are alive and there is no evidence for progression of disease. The age at diagnosis in the group with benign course was lower (39, 40, 43 years) than that in the group that recurred (48, 53 years). Both recurrent SMTLMs expressed less PR and, only quantitatively, more p53. One case expressed more cyclin D1 when compared to its primary. p16 level was reduced in one recurrent SMTLM. FISH of both SMTLMs that recurred revealed homozygous deletion of p53 and Rb loci in one of each primary and recurrent tumor. Loss of 1p36 loci was found in both recurrent SMTLMs, and in one case, this genetic imbalance was already present in the primary tumor. The current data provide evidence that the age at diagnosis of SMTLM is a prognostic factor and that LMS originating from SMTLM carries an overall better prognosis than primary LMS. Recurrent tumors showed decreased PR levels. Accumulation of genetic losses, including p53, Rb, and 1p36 loci, may identify a subgroup of SMTLMs with a higher potential towards malignancy.

Establishment and characterization of a cytogenetically complex Chinese multiple myeloma-derived cell line with homozygous p53 deletion and cyclin E overexpression

We describe the establishment and characterization of a new myeloma-derived cell line (MM17), originating from the sacral plasmacytoma of a 54-year-old Chinese woman diagnosed with multiple myeloma (MM). MM17 was confirmed morphologically and immunophenotypically to be clonal plasma cells positive for CD38 and CD138 and negative for EBV marker. Authenticity was confirmed using comparative genomic hybridization and DNA fingerprinting studies on bone marrow aspirate, sacral tumor tissue and MM17. Combined G-banding and multicolor fluorescence in situ hybridization analyses demonstrated a primarily hypodiploid karyotype with two sidelines sharing common stemline aberrations: +6, -7, -10, -13, -14, -17, -X, der(1;17)(q10;q10), t(2;7)(q23;q11.2), t(8;14)(q24;q32) and ins(16;1)(q13;?q22q41); and a number of hypertriploid cells. The involvement of p53 alteration and cyclin E overexpression, both with relevance to the induction of chromosomal instability, was investigated in MM17 and together with two other MM derived cell lines (U266 and IM-9) for cyclin E expression. Homozygous deletion of p53 gene hitherto not reported in MM, was detected. Both MM17 and U266 with complex cytogenetic aberrations demonstrated overexpression of cyclin E1 and E2, whereas IM-9 with a normal karyotype showed cyclin E2 but not E1 overexpression. These data suggested that E1 but not E2 overexpression was associated with chromosomal abnormalities observed in MM17 and U266, which provides the first supporting evidence for the link of cyclin E and chromosomal instability in MM. This is the first characterized Chinese MM-derived cell line with homozygous p53 deletion which may serve as a valuable in vitro system for studying MM pathogenesis particularly for Chinese.

Inactivation of p53 tumor suppressor gene acts synergistically with c-neu oncogene in salivary gland tumorigenesis

Transgenic mice expressing specific oncogenes usually develop tumors in a stochastic fashion suggesting that tumor progression is a multi-step process. To gain further understanding of the interactions between oncogenes and tumor suppressor genes during tumorigenesis, we have crossed a transgenic strain (TG.NK) carrying an activated c-neu oncogene driven by the MMTV enhancer/promoter with p53-deficient mice. c-neu transgenic mice have stochastic breast tumor formation and normal appearing salivary glands. However, c-neu mice heterozygous for a p53 deletion develop parotid gland tumors and loose their wild type p53 allele. c-neu mice with a homozygous p53 deletion have increased rates of parotid tumor onset suggesting that inactivation of p53 is required and sufficient for parotid gland transformation in the presence of activated c-neu. In contrast to the dramatic effect of p53 in parotid gland transformation, p53 loss has little effect on the rate or stochastic appearance of mammary tumors. In addition, p53 loss was accompanied by the down regulation of p21 in parotid gland tumors but not breast tumors. The parotid gland tumors were aneuploid and demonstrated increased levels of Cyclin D1 expression. These observations suggest that in c-neu transgenic mice, p53 alterations have differential tissue effects and may be influenced by the tissue specific expression of genes influencing p53 activity.

Isolation by Size of Epithelial Tumor Cells: A New Method for the Immunomorphological and Molecular Characterization of Circulating Tumor Cells

We have developed a new assay, ISET (isolation by size of epithelial tumor cells), which allows the counting and the immunomorphological and molecular characterization of circulating tumor cells in patients with carcinoma, using peripheral blood sample volumes as small as 1 ml. Using this assay, epithelial tumor cells can be isolated individually by filtration because of their larger size when compared to peripheral blood leukocytes. ISET parameters were defined using peripheral blood spiked with tumor cell lines (HepG2, Hep3B, MCF-7, HeLa, and LNCaP). ISET can detect a single, micropipetted tumor cell, added to 1 ml of blood. We also demonstrate that fluorescence in situ hybridization can be used to perform chromosomal analyses on tumor cells collected using ISET. Polymerase chain reaction-based genetic analyses can be applied to ISET-isolated cells, and, as an example, we demonstrate homozygous p53 deletion in single Hep3B cells after filtration and laser microdissection. Finally, we provide evidence for the in vivo feasibility of ISET in patients with hepatocellular carcinoma undergoing tumor resection. ISET, but not reverse transcriptase-polymerase chain reaction, allowed analysis of cell morphology, counting of tumor cells, and demonstration of tumor microemboli spread into peripheral blood during surgery. Overall, ISET constitutes a novel approach that should open new perpectives in molecular medicine.