Mark J. Hoenerhoff

Bmi-1 Cooperates with H-Ras to Transform Human Mammary Epithelial Cells via Dysregulation of Multiple Growth-Regulatory Pathways

Elevated expression of Bmi-1 is associated with many cancers, including breast cancer. Here, we examined the oncogenic potential of Bmi-1 in MCF10A cells, a spontaneously immortalized, nontransformed strain of human mammary epithelial cells (HMEC). Bmi-1 overexpression alone in MCF10A cells did not result in oncogenic transformation. However, Bmi-1 co-overexpression with activated H-Ras (RasG12V) resulted in efficient transformation of MCF10A cells in vitro. Although early-passage H-Ras-expressing MCF10A cells were not transformed, late-passage H-Ras-expressing cells exhibited features of transformation in vitro. Early- and late-passage H-Ras-expressing cells also differed in levels of expression of H-Ras and Ki-67, a marker of proliferation. Subsets of early-passage H-Ras-expressing cells exhibited high Ras expression and were negative for Ki-67, whereas most late-passage H-Ras-expressing cells expressed low levels of Ras and were Ki-67 positive. Injection of late-passage H-Ras-expressing cells in severe combined immunodeficient mice formed carcinomas with leiomatous, hemangiomatous, and mast cell components; these tumors were quite distinct from those induced by late-passage cells co-overexpressing Bmi-1 and H-Ras, which formed poorly differentiated carcinomas with spindle cell features. Bmi-1 and H-Ras co-overexpression in MCF10A cells also induced features of epithelial-to-mesenchymal transition. Importantly, Bmi-1 inhibited senescence and permitted proliferation of cells expressing high levels of Ras. Examination of various growth-regulatory pathways suggested that Bmi-1 overexpression together with H-Ras promotes HMEC transformation and breast oncogenesis by deregulation of multiple growth-regulatory pathways by p16(INK4a)-independent mechanisms.

BMI1 Cooperates with H-RAS to Induce an Aggressive Breast Cancer Phenotype with Brain Metastases

B-lymphoma Moloney murine leukaemia virus insertion region-1 (BMI1) is a member of the polycomb group of transcription repressors, which functions in stem cell maintenance and oncogenesis through the inhibition of the INK4A/ARF tumour suppressor locus. Overexpression of BMI1 is associated with poor prognosis in several human cancers, including breast cancer. We have previously shown that BMI1 collaborates with H-RAS to induce transformation of MCF10A human mammary epithelial cells through dysregulation of multiple growth pathways independent of the INK4A/ARF locus. In this study, we show that BMI1 collaborates with H-RAS to promote increased proliferation, invasion and resistance to apoptosis in vitro, and an increased rate of spontaneous metastases from mammary fat pad xenografts including novel metastases to the brain. Furthermore, in collaboration with H-RAS, BMI1 induced fulminant metastatic disease in the lung using a tail vein model of haematogenous spread through accelerated cellular proliferation and inhibition of apoptosis. Finally, we show that knockdown of BMI1 in several established breast cancer cell lines leads to decreased oncogenic behaviour in vitro and in vivo. In summary, BMI1 collaborates with H-RAS to induce an aggressive and metastatic phenotype with the unusual occurrence of brain metastasis, making it an important target for diagnosis and treatment of aggressive breast cancer.

Identification of an integrated SV40 T/t-antigen cancer signature in aggressive human breast, prostate, and lung carcinomas with poor prognosis

Understanding the genetic architecture of cancer pathways that distinguishes subsets of human cancer is critical to developing new therapies that better target tumors based on their molecular expression profiles. In this study, we identify an integrated gene signature from multiple transgenic models of epithelial cancers intrinsic to the functions of the Simian virus 40 T/t-antigens that is associated with the biological behavior and prognosis for several human epithelial tumors. This genetic signature, composed primarily of genes regulating cell replication, proliferation, DNA repair, and apoptosis, is not a general cancer signature. Rather, it is uniquely activated primarily in tumors with aberrant p53, Rb, or BRCA1 expression but not in tumors initiated through the overexpression of myc, ras, her2/neu, or polyoma middle T oncogenes. Importantly, human breast, lung, and prostate tumors expressing this set of genes represent subsets of tumors with the most aggressive phenotype and with poor prognosis. The T/t-antigen signature is highly predictive of human breast cancer prognosis. Because this class of epithelial tumors is generally intractable to currently existing standard therapies, this genetic signature identifies potential targets for novel therapies directed against these lethal forms of cancer. Because these genetic targets have been discovered using mammary, prostate, and lung T/t-antigen mouse cancer models, these models are rationale candidates for use in preclinical testing of therapies focused on these biologically important targets.

Combined SFK/MEK inhibition prevents metastatic outgrowth of dormant tumor cells

Breast cancer (BC) can recur as metastatic disease many years after primary tumor removal, suggesting that disseminated tumor cells survive for extended periods in a dormant state that is refractory to conventional therapies. We have previously shown that altering the tumor microenvironment through fibrosis with collagen and fibronectin deposition can trigger tumor cells to switch from a dormant to a proliferative state. Here, we used an in vivo preclinical model and a 3D in vitro model of dormancy to evaluate the role of Src family kinase (SFK) in regulating this dormant-to-proliferative switch. We found that pharmacological inhibition of SFK signaling or Src knockdown results in the nuclear localization of cyclin-dependent kinase inhibitor p27 and prevents the proliferative outbreak of dormant BC cells and metastatic lesion formation; however, SFK inhibition did not kill dormant cells. Dormant cell proliferation also required ERK1/2 activation. Combination treatment of cells undergoing the dormant-to-proliferative switch with the Src inhibitor (AZD0530) and MEK1/2 inhibitor (AZD6244) induced apoptosis in a large fraction of the dormant cells and delayed metastatic outgrowth, neither of which was observed with either inhibitor alone. Thus, targeting Src prevents the proliferative response of dormant cells to external stimuli, but requires MEK1/2 inhibition to suppress their survival. These data indicate that treatments targeting Src in combination with MEK1/2 may prevent BC recurrence.

A Review of the Molecular Mechanisms of Chemically-Induced Neoplasia in Rat and Mouse Models in National Toxicology Program Bioassays and Their Relevance to Human Cancer

Tumor response in the B6C3F1 mouse, F344 rat, and other animal models following exposure to various compounds provides evidence that people exposed to these or similar compounds may be at risk for developing cancer. Although tumors in rodents and humans are often morphologically similar, underlying mechanisms of tumorigenesis are often unknown and may be different between the species. Therefore, the relevance of an animal tumor response to human health would be better determined if the molecular pathogenesis were understood. The underlying molecular mechanisms leading to carcinogenesis are complex and involve multiple genetic and epigenetic events and other factors. To address the molecular pathogenesis of environmental carcinogens, the authors examine rodent tumors (e.g., lung, colon, mammary gland, skin, brain, mesothelioma) for alterations in cancer genes and epigenetic events that are associated with human cancer. National Toxicology Program (NTP) studies have identified several genetic alterations in chemically induced rodent neoplasms that are important in human cancer. Identification of such alterations in rodent models of chemical carcinogenesis caused by exposure to environmental contaminants, occupational chemicals, and other compounds lends further support that they are of potential human health risk. These studies also emphasize the importance of molecular evaluation of chemically induced rodent tumors for providing greater public health significance for NTP evaluated compounds.

GATA3 inhibits lysyl oxidase-mediated metastases of human basal triple-negative breast cancer cells

Discovery of mechanisms that impede the aggressive and metastatic phenotype of human basal triple-negative-type breast cancers (BTNBCs) could provide novel targets for therapy for this form of breast cancer that has a relatively poor prognosis. Previous studies have demonstrated that expression of GATA3, the master transcriptional regulator of mammary luminal differentiation, can reduce the tumorigenicity and metastatic propensity of the human BTNBC MDA-MB-231 cell line (MB231), although the mechanism for reduced metastases was not elucidated. We demonstrate through gene expression profiling that GATA3 expression in 231 cells resulted in the dramatic reduction in the expression of lysyl oxidase (LOX), a metastasis-promoting, matrix-remodeling protein, in part, through methylation of the LOX promoter. Suppression of LOX expression by GATA3 was further confirmed in the BTNBC Hs578T cell line. Conversely, reduction of GATA3 expression by small interfering RNA in luminal BT474 cells increased LOX expression. Reconstitution of LOX expression in 231-GATA3 cells restored metastatic propensity. A strong inverse association between LOX and GATA3 expression was confirmed in a panel of 51 human breast cancer cell lines. Similarly, human breast cancer microarray data demonstrated that high LOX/low GATA3 expression is associated with the BTNBC subtype of breast cancer and poor patient prognosis. Expression of GATA3 reprograms BTNBCs to a less aggressive phenotype and inhibits a major mechanism of metastasis through inhibition of LOX. Induction of GATA3 in BTNBC cells or novel approaches that inhibit LOX expression or activity could be important strategies for treating BTNBCs.

Global Gene Profiling of Spontaneous Hepatocellular Carcinoma in B6C3F1 Mice: Similarities in the Molecular Landscape with Human Liver Cancer

Hepatocellular carcinoma (HCC) is an important cause of morbidity and mortality worldwide. Although the risk factors of human HCC are well known, the molecular pathogenesis of this disease is complex, and in general, treatment options remain poor. The use of rodent models to study human cancer has been extensively pursued, both through genetically engineered rodents and rodent models used in carcinogenicity and toxicology studies. In particular, the B6C3F1 mouse used in the National Toxicology Program (NTP) two-year bioassay has been used to evaluate the carcinogenic effects of environmental and occupational chemicals, and other compounds. The high incidence of spontaneous HCC in the B6C3F1 mouse has challenged its use as a model for chemically induced HCC in terms of relevance to the human disease. Using global gene expression profiling, we identify the dysregulation of several mediators similarly altered in human HCC, including re-expression of fetal oncogenes, upregulation of protooncogenes, downregulation of tumor suppressor genes, and abnormal expression of cell cycle mediators, growth factors, apoptosis regulators, and angiogenesis and extracellular matrix remodeling factors. Although major differences in etiology and pathogenesis remain between human and mouse HCC, there are important similarities in global gene expression and molecular pathways dysregulated in mouse and human HCC. These data provide further support for the use of this model in hazard identification of compounds with potential human carcinogenicity risk, and may help in better understanding the mechanisms of tumorigenesis resulting from chemical exposure in the NTP two-year carcinogenicity bioassay.

Hepatocellular Carcinomas in B6C3F1 Mice Treated with Ginkgo biloba Extract for Two Years Differ from Spontaneous Liver Tumors in Cancer Gene Mutations and Genomic Pathways

Ginkgo biloba leaf extract (GBE) has been used for centuries in traditional Chinese medicine and today is used as an herbal supplement touted for improving neural function and for its antioxidant and anticancer effects. Herbal supplements have the potential for consumption over extended periods of time, with a general lack of sufficient data on long-term carcinogenicity risk. Exposure of B6C3F1 mice to GBE in the 2-year National Toxicology Program carcinogenicity bioassay resulted in a dose-dependent increase in hepatocellular tumors, including hepatocellular carcinoma (HCC). We show that the mechanism of hepatocarcinogenesis in GBE exposed animals is complex, involving alterations in H-ras and Ctnnb1 mutation spectra, WNT pathway dysregulation, and significantly altered gene expression associated with oncogenesis, HCC development, and chronic xenobiotic and oxidative stress compared to spontaneous HCC. This study provides a molecular context for the genetic changes associated with hepatocarcinogenesis in GBE exposed mice and illustrates the marked differences between these tumors and those arising spontaneously in the B6C3F1 mouse. The molecular changes observed in HCC from GBE-treated animals may be of relevance to those seen in human HCC and other types of cancer, and provide important data on potential mechanisms of GBE hepatocarcinogenesis.

Proceedings of the 2012 National Toxicology Program Satellite Symposium.

The 2012 annual National Toxicology Program (NTP) Satellite Symposium, entitled “Pathology Potpourri,” was held in Boston in advance of the Society of Toxicologic Pathologys 31st annual meeting. The goal of the NTP Symposium is to present current diagnostic pathology or nomenclature issues to the toxicologic pathology community. This article presents summaries of the speakers’ presentations, including diagnostic or nomenclature issues that were presented, along with select images that were used for audience voting or discussion. Some lesions and topics covered during the symposium include eosinophilic crystalline pneumonia in a transgenic mouse model; differentiating adrenal cortical cystic degeneration from adenoma; atypical eosinophilic foci of altered hepatocytes; differentiating cardiac schwannoma from cardiomyopathy; diagnosis of cardiac papillary muscle lesions; intrahepatocytic erythrocytes and venous subendothelial hepatocytes; lesions in Rathke’s cleft and pars distalis; pernicious anemia and megaloblastic disorders; embryonic neuroepithelial dysplasia, holoprosencephaly and exencephaly; and INHAND nomenclature for select cardiovascular lesions.

Evaluation of dosages and routes of administration of tramadol analgesia in rats using hot-plate and tail-flick tests.

Tramadol is an opioid-like analgesic with relatively mild side effects. Because it is inexpensive and is not classified as a controlled substance by the US federal government, the authors wanted to evaluate its applicability as a practical and effective analgesic in male Sprague Dawley rats. They measured the efficacy of four dosages (4, 12.5, 25 or 50 mg tramadol per kg body weight) and three routes of administration (per os (p.o.) in a flavored gelatin cube, subcutaneous (s.c.) or intraperitoneal (i.p.)) using the hot-plate test and the tail-flick test, which were carried out 1 week apart. Rats that were dosed p.o. were given flavored gelatin cubes without tramadol on the 2 d before testing to help them become acclimated to the gelatin, in an effort to increase the likelihood that they would consume the gelatin on the testing day. Results from the hot-plate and tail-flick tests for rats that were given tramadol p.o. were similar before and after administration, regardless of tramadol dosage, suggesting that this route of administration was not effective. The s.c. route of administration was effective at dosages of 25 mg and 50 mg tramadol per kg body weight, although these dosages also resulted in sedation and skin lesions. The i.p. route of administration was also effective at dosages of 12.5 mg, 25 mg and 50 mg tramadol per kg body weight, though sedation was observed at dosages of 25 mg and 50 mg per kg body weight. Intraperitoneal administration of 12.5 mg tramadol per kg body weight had no notable side effects, and the authors plan to further study this dosage and route of administration in a rodent surgical model of pain.