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Claudin-low breast cancer represents an aggressive molecular subtype that is comprised of mostly triple-negative mammary tumor cells that possess stem cell-like and mesenchymal features. Little is known about the cellular origin and oncogenic drivers that promote claudin-low breast cancer. In this study, we show that persistent oncogenic RAS signaling causes highly metastatic triple-negative mammary tumors in mice. More importantly, the activation of endogenous mutant KRAS and expression of exogenous KRAS specifically in luminal epithelial cells in a continuous and differentiation stage-independent manner induces preneoplastic lesions that evolve into basal-like and claudin-low mammary cancers. Further investigations demonstrate that the continuous signaling of oncogenic RAS, as well as regulators of EMT, play a crucial role in the cellular plasticity and maintenance of the mesenchymal and stem cell characteristics of claudin-low mammary cancer cells., The cellular origin and oncogenic drivers promoting claudin-low breast cancer are undefined. Here, the authors report that the consistent activation of oncogenic RAS signaling, as well as regulators of EMT, play a crucial role in the cellular plasticity and maintenance of the mesenchymal and stem cell characteristics of claudin-low mammary cancer cells.

Recent genome analysis of human prostate cancers demonstrated that both AR gene amplification and TP53 mutation are among the most frequently observed alterations in advanced prostate cancer. However, the biological role of these dual genetic alterations in prostate tumorigenesis is largely unknown. In addition, there are no biologically relevant models that can be used to assess the molecular mechanisms for these genetic abnormalities. Here, we report a novel mouse model, in which elevated transgenic AR expression and Trp53 deletion occur simultaneously in mouse prostatic epithelium to mimic human prostate cancer cells. These compound mice developed an earlier onset of high-grade prostatic intraepithelial neoplasia and accelerated prostate tumors in comparison with mice harboring only the AR transgene. Histological analysis showed prostatic sarcomatoid and basaloid carcinomas with massive squamous differentiation in the above compound mice. RNA-sequencing analyses identified a robust enrichment of the signature genes for human prostatic basal cell carcinomas in the above prostate tumors. Master regulator analysis revealed SOX2 as a transcriptional regulator in prostatic basal cell tumors. Elevated expression of SOX2 and its downstream target genes were detected in prostatic tumors of the compound mice. Chromatin immunoprecipitation analyses implicate a coregulatory role of AR and SOX2 in the expression of prostatic basal cell signature genes. Our data demonstrate a critical role of SOX2 in prostate tumorigenesis and provide mechanistic insight into prostate tumor aggressiveness and progression mediated by aberrant AR and p53 signaling pathways.

HER2-positive breast cancers are among the most heterogeneous breast cancer subtypes. The early amplification of HER2 and its known oncogenic isoforms provide a plausible mechanism in which distinct programs of tumor heterogeneity could be traced to the initial oncogenic event. Here a Cancer rainbow mouse simultaneously expressing fluorescently barcoded wildtype (WTHER2), exon-16 null (d16HER2), and N-terminally truncated (p95HER2) HER2 isoforms is used to trace tumorigenesis from initiation to invasion. Tumorigenesis was visualized using whole-gland fluorescent lineage tracing and single-cell molecular pathology. We demonstrate that within weeks of expression, morphologic aberrations were already present and unique to each HER2 isoform. Although WTHER2 cells were abundant throughout the mammary ducts, detectable lesions were exceptionally rare. In contrast, d16HER2 and p95HER2 induced rapid tumor development. d16HER2 incited homogenous and proliferative luminal-like lesions which infrequently progressed to invasive phenotypes whereas p95HER2 lesions were heterogenous and invasive at the smallest detectable stage. Distinct cancer trajectories were observed for d16HER2 and p95HER2 tumors as evidenced by oncogene-dependent changes in epithelial specification and the tumor microenvironment. These data provide direct experimental evidence that intratumor heterogeneity programs begin very early and well in advance of screen or clinically detectable breast cancer. Implications: Although all HER2 breast cancers are treated equally, we show a mechanism by which clinically undetected HER2 isoforms program heterogenous cancer phenotypes through biased epithelial specification and adaptations within the tumor microenvironment.

During puberty, a woman’s breasts are vulnerable to environmental damage (“window of vulnerability”). Early exposure to environmental carcinogens, endocrine disruptors, and unhealthy foods (refined sugar, processed fats, food additives) are hypothesized to promote molecular damage that increases breast cancer risk. However, prospective human studies are difficult to perform and effective interventions to prevent these early exposures are lacking. It is difficult to prevent environmental exposures during puberty. Specifically, young women are repeatedly exposed to media messaging that promotes unhealthy foods. Young women living in disadvantaged neighborhoods experience additional challenges including a lack of access to healthy food and exposure to contaminated air, water, and soil. The purpose of this review is to gather information on potential exposures during puberty. In future directions, this information will be used to help elementary/middle-school girls to identify and quantitate environmental exposures and develop cost-effective strategies to reduce exposures.

Field cancerization is a premalignant process marked by clones of oncogenic mutations spreading through the epithelium. The timescales of intestinal field cancerization can be variable and the mechanisms driving the rapid spread of oncogenic clones are unknown. Here we use a Cancer rainbow (Crainbow) modelling system for fluorescently barcoding somatic mutations and directly visualizing the clonal expansion and spread of oncogenes. Crainbow shows that mutations of ß-catenin (Ctnnb1) within the intestinal stem cell results in widespread expansion of oncogenes during perinatal development but not in adults. In contrast, mutations that extrinsically disrupt the stem cell microenvironment can spread in adult intestine without delay. We observe the rapid spread of premalignant clones in Crainbow mice expressing oncogenic Rspondin-3 (RSPO3), which occurs by increasing crypt fission and inhibiting crypt fixation. Crainbow modelling provides insight into how somatic mutations rapidly spread and a plausible mechanism for predetermining the intratumor heterogeneity found in colon cancers., Pre-malignant cells harbouring oncogenic mutations can populate and spread throughout a tissue. Here, using a rainbow mouse system, the authors explore how clonal expansion in the mouse intestine might explain high levels of intra-tumoural heterogeneity observed in the disease.

The receptor tyrosine kinase Ret, a key gain-of-function mutated oncoprotein in thyroid carcinomas, has recently been implicated in other cancer types. While Ret copy number gains and mutations have been reported at low frequencies in breast tumors, we and others have reported that Ret is overexpressed in about 40% of human tumors and this correlates with poor patient prognosis. Ret activation regulates numerous intracellular pathways related to proliferation and inflammation, but it is not known whether abnormal Ret expression is sufficient to induce mammary carcinomas. Using a novel doxycycline-inducible transgenic mouse model with the MMTV promoter controlling Ret expression, we show that overexpression of wild-type Ret in the mammary epithelium produces mammary tumors, displaying a morphology that recapitulates characteristics of human luminal breast tumors. Ret-evoked tumors are estrogen receptor positive and negative for progesterone receptor. Moreover, tumors rapidly regress after doxycycline withdrawal, indicating that Ret is the driving oncoprotein. Using next-generation sequencing, we examined the levels of transcripts in these tumors, confirming a luminal signature. Ret-evoked tumors have been passaged in mice and used to test novel therapeutic approaches. Importantly, we have determined that tumors are resistant to endocrine therapy, but respond successfully to treatment with a Ret kinase inhibitor. Our data provide the first compelling evidence for an oncogenic role of non-mutated Ret in the mammary gland and are an incentive for clinical development of Ret as a cancer biomarker and therapeutic target.

Triple-negative breast cancer (TNBC) commonly develops resistance to chemotherapy, yet markers predictive of chemoresistance in this disease are lacking. Here, we define WNT10B-dependent biomarkers for β-CATENIN/HMGA2/EZH2 signaling predictive of reduced relapse-free survival. Concordant expression of HMGA2 and EZH2 proteins is observed in MMTV-Wnt10bLacZ transgenic mice during metastasis, and Hmga2 haploinsufficiency decreased EZH2 protein expression, repressing lung metastasis. A novel autoregulatory loop interdependent on HMGA2 and EZH2 expression is essential for β-CATENIN/TCF-4/LEF-1 transcription. Mechanistically, both HMGA2 and EZH2 displaced Groucho/TLE1 from TCF-4 and served as gatekeepers for K49 acetylation on β-CATENIN, which is essential for transcription. In addition, we discovered that HMGA2-EZH2 interacts with the PRC2 complex. Absence of HMGA2 or EZH2 expression or chemical inhibition of Wnt signaling in a chemoresistant patient-derived xenograft (PDX) model of TNBC abolished visceral metastasis, repressing AXIN2, MYC, EZH2, and HMGA2 expression in vivo. Combinatorial therapy of a WNT inhibitor with doxorubicin synergistically activated apoptosis in vitro, resensitized PDX-derived cells to doxorubicin, and repressed lung metastasis in vivo. We propose that targeting the WNT10B biomarker network will provide improved outcomes for TNBC. Significance: These findings reveal targeting the WNT signaling pathway as a potential therapeutic strategy in triple-negative breast cancer.

Loss of normal development is a hallmark of cancer. Thus, understanding the mechanisms of tissue-specific developmental regulation and the changes that occur during tumorigenesis may provide insights of both diagnostic and therapeutic importance. In breast cancer, several members of the receptor tyrosine kinases (RTK) family that are well known to promote aggressive breast cancers also have roles in normal breast. We found that Ret, a RTK member, is normally expressed in the mouse glands in lactation. We determined that inhibition of Ret activity in vivo does not alter lactation, however impacts in the transition to involution. Involution is the period with high inflammation which returns the lactating mammary gland to a quiescent state after weaning. Involution has been well described as a post-lactation stage that drives cancer progression. Ret is overexpressed in about 40% of human breast tumors. Previously, using a doxycycline-inducible transgenic mouse model (Ret/MTB) we determined that chronic expression of Ret is oncogenic in the mammary epithelium. However, the stage of development at which Ret expression results in increase mammary tumor incidence has not been identified. To address this, we used the Ret/MTB system, to conditionally overexpress Ret during discrete periods of mammary gland development. We found that Ret is required for efficient transition to involution. We determined that the induction of Ret in Ret/MTB females promotes the expression of factors that drives involution, including premature Stat3 activation. RNA-seq data in Ret-overexpressing glands is supporting these findings, which were confirmed by several techniques. In addition, sustained expression of Ret during post-lactation enhances cancer potential showing a significant increase in pre-neoplastic lesions, defective milk recycling and disrupting Stat3 signaling. These results demonstrate that Ret deregulation increases cancer potential in post-lactation and might be considered as a prognostic marker for post-partum breast cancer. Citation Format: Sabrina A. Vallone, Martín García Solá, Robert D. Cardiff, Roberto P. Meiss, Lewis A. Chodosh, Carolina Shere-Levy, Edith C C. Kordon, Nancy E. Hynes, Albana Gattelli. Sustained Ret expression during mammary gland post-lactation induces premature involution and enhances cancer potential [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3685.

The contribution of exposure to the persistent, lipophilic pesticide dichlorodiphenyltrichloroethane (DDT) to breast cancer risk is controversial. A recent report by the International Agency for Research on Cancer (IARC) concluded that no consistent association exists between DDT exposure and breast cancer, yet stressed that there was a lack of existing evidence to allow for the determination of the effects of early-life exposure to DDT. It has been hypothesized that exposure to DDT during critical periods of breast development may increase breast cancer risk. We hypothesized that perinatal DDT exposure would result in shortened latency of spontaneous mammary tumorigenesis and accelerated growth of mammary tumors and increase the incidence of lung metastasis. To test the effect on mammary tumor early progression and growth as well as lung metastasis, C57BL/6J female mice, pregnant as a result of mating with B6.FVB-Tg(MMTV-PyVT)634Mul/LellJ male mice, were administered DDT from gestational day 11.5 to postnatal day 5 to produce circulating levels of DDT in dams within the prenatal range associated with breast cancer in adult daughters. This perinatal DDT exposure significantly accelerated the development of palpable mammary tumors (hazard ratio = 3.7 (95% confidence interval 2.0-6.8; p Citation Format: Tomoko Ishikawa, Jason Tong, Alexander Borowsky, Robert Cardiff, Michele La Merrill. Perinatal DDT exposure shortens latency of mouse mammary tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A48.

Mouse models and genetically engineered mouse models (GEMM) are essential experimental tools for the understanding molecular mechanisms within complex biological systems. GEMM are especially useful for inferencing phenocopy information to genetic human diseases such as breast cancer. Human breast cancer modeling in mice most commonly employs mammary epithelial-specific promoters to investigate gene function(s) and, in particular, putative oncogenes. Models are specifically useful in the mammary epithelial cell in the context of the complete mammary gland environment. Gene targeted knockout mice including conditional targeting to specific mammary cells can reveal developmental defects in mammary organogenesis and demonstrate the importance of putative tumor suppressor genes. Some of these models demonstrate a non-traditional type of tumor suppression which involves interplay between the tumor susceptible cell and its host/environment. These GEMM help to reveal the processes of cancer progression beyond those intrinsic to cancer cells. Furthermore, the, analysis of mouse models requires appropriate consideration of mouse strain, background, and environmental factors. In this review, we compare aging-related factors in mouse models for breast cancer. We introduce databases of GEMM attributes and colony functional variations.

The tumor suppressor TP53 can initiate a plethora of anti-proliferative effects to maintain genomic integrity under conditions of genotoxic stress. The N-terminal proline-rich domain (PRD) of TP53 is important in the regulation of TP53 activity and stability. A common polymorphism at codon 72 in this region has been associated with altered cancer risk in humans. The Trp53ΔP mouse, which carries a germline homozygous deletion of a region of the PRD, does not develop spontaneous tumors in a mixed 129/Sv and C57BL/6 genetic background, but is highly susceptible to a broad range of tumor types following total body exposure to 4 Gy gamma (γ) radiation. This contrasts with the tumor spectrum in Trp53 null (-/-) mice, which mainly develop thymic lymphomas and osteosarcomas. Analysis of genomic instability in tissues and cells from Trp53ΔP mice demonstrated elevated basal levels of aneuploidy, but this is not sufficient to drive spontaneous tumorigenesis, which requires an additional DNA damage stimulus. Levels of genomic instability did not increase significantly in Trp53ΔP mice following irradiation exposure, suggesting that other radiation effects including tissue inflammation, altered metabolism or autophagy, may play an important role. The Trp53ΔP mouse is a novel model to dissect the mechanisms of tumor development induced by radiation exposure. © 2015 Wiley Periodicals, Inc.

Microwave methods of fixation can dramatically shorten fixation times while preserving tissue structure; however, it remains unclear if adequate tissue antigenicity is preserved. To assess and validate antigenicity, robust quantitative methods and animal disease models are needed. We used two mouse mammary models of human breast cancer to evaluate microwave-assisted and standard 24-hr formalin fixation. The mouse models expressed four antigens prognostic for breast cancer outcome: estrogen receptor, progesterone receptor, Ki67, and human epidermal growth factor receptor 2. Using pathologist evaluation and novel methods of quantitative image analysis, we measured and compared the quality of antigen preservation, percentage of positive cells, and line plots of cell intensity. Visual evaluations by pathologists established that the amounts and patterns of staining were similar in tissues fixed by the different methods. The results of the quantitative image analysis provided a fine-grained evaluation, demonstrating that tissue antigenicity is preserved in tissues fixed using microwave methods. Evaluation of the results demonstrated that a 1-hr, 150-W fixation is better than a 45-min, 150-W fixation followed by a 15-min, 650-W fixation. The results demonstrated that microwave-assisted formalin fixation can standardize fixation times to 1 hr and produce immunohistochemistry that is in every way commensurate with longer conventional fixation methods.

E-cadherin complexes with the actin cytoskeleton via cytoplasmic catenins and maintains the functional characteristics and integrity of the epithelia in normal epithelial tissues. Lost expression of E-cadherin disrupts this complex resulting in loss of cell polarity, epithelial denudation and increased epithelial permeability in a variety of tissues. Decreased expression of E-cadherin has also been observed in invasive and metastatic human tumors. In this study, we investigated the effect of E-cadherin loss in prostatic epithelium using newly developed genetically engineered mouse models. Deletion of E-cadherin in prostatic luminal epithelial cells with modified probasin promoter driven Cre (PB-Cre4) induced the development of mouse prostatic intraepithelial neoplasia (PIN). An increase in levels of cytoplasmic and nuclear β-catenin appeared in E-cadherin deleted atypical cells within PIN lesions. Using various experimental approaches, we further demonstrated that the knockdown of E-cadherin expression elevated free cytoplasmic and nuclear β-catenin and enhanced androgen-induced transcription and cell growth. Intriguingly, pathological changes representing prostatic epithelial cell denudation and increased apoptosis accompanied the above PIN lesions. The essential role of E-cadherin in maintaining prostatic epithelial integrity and organization was further demonstrated using organoid culture approaches. To directly assess the role of loss of E-cadherin in prostate tumor progression, we generated a new mouse model with bigenic Cdh1 and Pten deletion in prostate epithelium. Early onset, aggressive tumor phenotypes presented in the compound mice. Strikingly, goblet cell metaplasia was observed, intermixed within prostatic tumor lesions of the compound mice. This study provides multiple lines of novel evidence demonstrating a comprehensive role of E-cadherin in maintaining epithelial integrity during the course of prostate oncogenic transformation, tumor initiation and progression., Author summary The biological significance of E-cadherin in maintaining prostatic epithelial integrity and related molecular mechanisms are still unclear. In this study, using mouse genetic tools, we directly address this important and unresolved question. Conditional deletion of E-cadherin in mouse prostatic epithelia resulted in prostatic intraepithelial neoplasia (PIN) development but no prostatic tumor formation. Both in vivo and in vitro data showed that loss of E-cadherin modulates the cellular localization of β-catenin, elevates its cytoplasmic and nuclear levels, and enhances its activity in transcription and cell proliferation. Intriguingly, in addition to PIN lesions, increased epithelial denudation and cell apoptosis also appeared within PIN lesions. This implicates that although lost E-cadherin is sufficient to introduce oncogenic transformation in prostatic epithelia, it also induces cell apoptosis and disrupts epithelial structure, preventing atypical PIN cells from progressing to tumor cells. Simultaneous deletion of Pten, a tumor suppressor, and E-cadherin in prostatic epithelia resulted in early onset, invasive prostatic tumors with admixture of goblet cells. These results demonstrate a critical role of E-cadherin in promoting prostatic tumor transdifferentiation and progression. This study further elucidates the dynamic role of E-cadherin in maintaining prostatic epithelial integrity during tumor initiation and progression.

This abstract was withdrawn by the authors. Citation Format: Penzvalto Z, Chen JQ, Cardiff RD, Willis B, Hubbard NE, Piersigilli A, Borowsky AD. Withdrawn [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-03-03.

The tumor suppressor p16Ink4a, encoded by the INK4a gene, is an inhibitor of cyclin D-dependent kinases 4 and 6, CDK4 and CDK6. This inhibition prevents the phosphorylation of the retinoblastoma protein (pRb), resulting in cellular senescence through inhibition of E2F-mediated transcription of S phase genes required for cell proliferation. The p16Ink4a plays an important role in tumor suppression, whereby its deletion, mutation, or epigenetic silencing is a frequently observed genetic alteration in prostate cancer. To assess its roles and related molecular mechanisms in prostate cancer initiation and progression, we generated a mouse model with conditional deletion of p16Ink4a in prostatic luminal epithelium. The mice underwent oncogenic transformation and developed prostatic intraepithelial neoplasia (PIN) from eight months of age, but failed to develop prostatic tumors. Given the prevalence of aberrant androgen signaling pathways in prostate cancer initiation and progression, we then generated R26hARL/wt:p16L/L: PB-Cre4 compound mice, in which conditional expression of the human AR transgene and deletion of p16Ink4a co-occur in prostatic luminal epithelial cells. While R26hARL/wt:PB-Cre4 mice showed no visible pathological changes, R26hARL/wt:p16L/L: PB-Cre4 compound mice displayed an early onset of high-grade PIN (HGPIN), prostatic carcinoma, and metastatic lesions. Strikingly, we observed tumors resembling human sarcomatoid carcinoma with intermixed focal regions of signet ring cell carcinoma (SRCC) in the prostates of the compound mice. Further characterization of these tumors showed they were of luminal epithelial cell origin, and featured characteristics of epithelial to mesenchymal transition (EMT) with enhanced proliferative and invasive capabilities. Our results not only implicate a biological role for AR expression and p16Ink4a deletion in the pathogenesis of prostatic SRCC, but also provide a new and unique genetically engineered mouse (GEM) model for investigating the molecular mechanisms for SRCC development.

A key issue in cancer biology is whether oncogenic transformation of different cell types of origin within an adult tissue gives rise to distinct tumour subtypes that differ in their prognosis and/or treatment response. We now show that initiation of prostate tumours in basal or luminal epithelial cells in mouse models results in tumours with distinct molecular signatures that are predictive of human patient outcomes. Furthermore, our analysis of untransformed basal cells reveals an unexpected assay dependence of their stem cell properties in sphere formation and transplantation assays versus genetic lineage tracing during prostate regeneration and adult tissue homeostasis. Although oncogenic transformation of basal cells gives rise to tumours with luminal phenotypes, cross-species bioinformatic analyses indicate that tumours of luminal origin are more aggressive than tumours of basal origin, and identify a molecular signature associated with patient outcome. Our results reveal the inherent plasticity of basal cells, and support a model in which different cells of origin generate distinct molecular subtypes of prostate cancer.

Amplification and overexpression of erbB2/neu proto-oncogene is observed in 20-30% human breast cancer and is inversely correlated with the survival of the patient. Despite this, somatic activating mutations within erbB2 in human breast cancers are rare. However, we have previously reported that a splice isoform of erbB2, containing an in-frame deletion of exon 16 (herein referred to as ErbB2ΔEx16), results in oncogenic activation of erbB2 because of constitutive dimerization of the ErbB2 receptor. Here, we demonstrate that the ErbB2ΔEx16 is a major oncogenic driver in breast cancer that constitutively signals from the cell surface. We further show that inducible expression of the ErbB2ΔEx16 variant in mammary gland of transgenic mice results in the rapid development of metastatic multifocal mammary tumors. Genetic and biochemical characterization of the ErbB2ΔEx16-derived mammary tumors exhibit several unique features that distinguish this model from the conventional ErbB2 ones expressing the erbB2 proto-oncogene in mammary epithelium. Unlike the wild-type ErbB2-derived tumors that express luminal keratins, ErbB2ΔEx16-derived tumors exhibit high degree of intratumoral heterogeneity co-expressing both basal and luminal keratins. Consistent with these distinct pathological features, the ErbB2ΔEx16 tumors exhibit distinct signaling and gene expression profiles that correlate with activation of number of key transcription factors implicated in breast cancer metastasis and cancer stem cell renewal.

Differential diagnosis and therapy of heterogeneous breast tumors poses a major clinical challenge. To address the need for a comprehensive, noninvasive strategy to define the molecular and functional profiles of tumors in vivo, we investigated a novel combination of metabolic PET and diffusion-weighted (DW)-MRI in the polyoma virus middle T antigen transgenic mouse model of breast cancer. The implementation of a voxelwise analysis for the clustering of intra- and intertumoral heterogeneity in this model resulted in a multiparametric profile based on [18F]Fluorodeoxyglucose ([18F]FDG)-PET and DW-MRI, which identified three distinct tumor phenotypes in vivo, including solid acinar, and solid nodular malignancies as well as cystic hyperplasia. To evaluate the feasibility of this approach for clinical use, we examined estrogen receptor-positive and progesterone receptor-positive breast tumors from five patient cases using DW-MRI and [18F]FDG-PET in a simultaneous PET/MRI system. The postsurgical in vivo PET/MRI data were correlated to whole-slide histology using the latter traditional diagnostic standard to define phenotype. By this approach, we showed how molecular, structural (microscopic, anatomic), and functional information could be simultaneously obtained noninvasively to identify precancerous and malignant subtypes within heterogeneous tumors. Combined with an automatized analysis, our results suggest that multiparametric molecular and functional imaging may be capable of providing comprehensive tumor profiling for noninvasive cancer diagnostics. Cancer Res; 76(18); 5512–22. ©2016 AACR.

A hallmark of human cancer is heterogeneity, reflecting the complex series of changes resulting in the activation of oncogenes coupled with inactivation of tumor suppressor genes. Breast cancer is no exception and indeed, many studies have revealed considerable complexity and heterogeneity in the population of primary breast tumors and substantial changes in a recurrent breast tumor that has acquired metastatic properties and drug resistance. We have made use of a Myc-inducible transgenic mouse model of breast cancer in which elimination of Myc activity following tumor development initially leads to a regression of a subset of tumors generally followed by de novo Myc-independent growth. We have observed that tumors that grow independent of Myc expression have gene profiles that are distinct from the primary tumors with characteristics indicative of an epithelial-mesenchymal transition (EMT) phenotype. Phenotypic analyses of Myc-independent tumors confirm the acquisition of an EMT phenotype suggested to be associated with invasive and migratory properties in human cancer cells. Further genomic analyses reveal mouse mammary tumors growing independent of myc have a higher probability of exhibiting a gene signature similar to that observed for human 'tumor-initiating' cells. Collectively, the data reveal genetic alterations that underlie tumor progression and an escape from Myc-dependent growth in a transgenic mouse model that can provide insights to what occurs in human cancers as they acquire drug resistance and metastatic properties.

Understanding the molecular pathways that contribute to the aggressive behavior of human cancers is a critical research priority. The SNF1/AMPK-related protein kinase Hunk is overexpressed in aggressive subsets of human breast, ovarian, and colon cancers. Analysis of Hunk(–/–) mice revealed that this kinase is required for metastasis of c-myc–induced mammary tumors but not c-myc–induced primary tumor formation. Similar to c-myc, amplification of the proto-oncogene HER2/neu occurs in 10%–30% of breast cancers and is associated with aggressive tumor behavior. By crossing Hunk(–/–) mice with transgenic mouse models for HER2/neu-induced mammary tumorigenesis, we report that Hunk is required for primary tumor formation induced by HER2/neu. Knockdown and reconstitution experiments in mouse and human breast cancer cell lines demonstrated that Hunk is required for maintenance of the tumorigenic phenotype in HER2/neu-transformed cells. This requirement is kinase dependent and resulted from the ability of Hunk to suppress apoptosis in association with downregulation of the tumor suppressor p27(kip1). Additionally, we find that Hunk is rapidly upregulated following HER2/neu activation in vivo and in vitro. These findings provide what we believe is the first evidence for a role for Hunk in primary tumorigenesis and cell survival and identify this kinase as an essential effector of the HER2/neu oncogenic pathway.

Chromatin coregulators are important factors in tumorigenesis and cancer progression. ANCCA is an AAA+ ATPase and a bromodomain-containing nuclear coactivator for the estrogen and androgen receptors that is crucial for assembly of chromatin-modifying complexes and proliferation of hormone-responsive cancer cells. In this study, we show that ANCCA is overexpressed in >70% of breast tumors and that its high protein level correlates well with tumor histologic grades (P < 0.0001), highlighting ANCCA as a prognostic factor for poor overall survival and disease recurrence. Strikingly, high-level ANCCA correlated with triple-negative tumors that represent highly aggressive disease. Analysis of ANCCA transcript levels in multiple expression profiles of breast cancer identified ANCCA as a common signature gene, indicating that elevated transcripts also strongly correlate with tumor metastasis and poor survival. Biological and mechanistic investigations revealed that ANCCA is crucial for proliferation and survival of triple-negative/basal-like cancer cells and that it controls the expression of B-Myb, histone methyltransferase EZH2, and an Rb-E2F core program for proliferation, along with a subset of key mitotic kinesins and cell survival genes (IRS2, VEGF, and Akt1). In particular, ANCCA overexpression correlated strongly with EZH2 in tumors. Our results suggest that ANCCA may integrate multiple oncogenic programs in breast cancer, serving in particular as a prognostic marker and a therapeutic target for triple-negative cancers. Cancer Res; 70(22); 9402–12. ©2010 AACR.

The phosphoinositide 3-kinase (PI3K) pathway regulates mammalian cell growth, survival, and motility and plays a major pathogenetic role in human prostate cancer (PCa). However, the oncogenic contributions downstream of the PI3K pathway made by mammalian target of rapamycin complex 1 (mTORC1)–mediated cell growth signal transduction in PCa have yet to be elucidated in detail. Here, we engineered constitutive mTORC1 activation in prostate epithelium by a conditional genetic deletion of tuberous sclerosis complex 1 (Tsc1), a potent negative regulator of mTORC1 signaling. Epithelial inactivation was not immediately tumorigenic, but Tsc1-deficient mice developed prostatic intraepithelial neoplasia (mPIN) in lateral and anterior prostates by 6 months of age, with increasing disease penetrance over time. Lateral prostate lesions in 16- to 22-month-old mutant mice progressed to two types of more advanced lesions, adenomatous gland forming lesion (Type 1) and atypical glands embedded in massively expanded reactive stroma (Type 2). Both Type 1 and Type 2 lesions contained multiple foci of microinvasive carcinoma. Epithelial neoplastic and atypical stromal lesions persisted despite 4 weeks of RAD001 chemotherapy. Rapalogue resistance was not due to AKT or extracellular signal-regulated kinase 1/2 activation. Expression of the homeobox gene Nkx3.1 was lost in Tsc1-deficient mPIN, and it cooperated with TSC1 loss in mPIN initiation in doubly mutant Tsc1:Nkx3.1 prostatic epithelial knockout mice. Thus, TSC1 inactivation distal to PI3K and AKT activation is sufficient to activate a molecular signaling cascade producing prostatic neoplasia and focal carcinogenesis. Cancer Res; 70(21); 8937–47. ©2010 AACR.

Repeated administration of chemotherapeutics is typically required for the effective treatment of highly aggressive tumors and often results in systemic toxicity. We have created a copper-doxorubicin complex within the core of liposomes and applied the resulting particle in multi-dose therapy. Copper and doxorubicin concentrations in the blood pool were similar at 24 hours (~40% of the injected dose) indicating stable circulation of the complex. Highly-quenched doxorubicin fluorescence remained in the blood pool over tens of hours, with fluorescence increasing only with the combination of liposome disruption and copper trans-chelation. At 48 hours after injection, doxorubicin fluorescence within the heart and skin was one-fifth and one-half, respectively, of fluorescence observed with ammonium sulfate-loaded doxorubicin liposomes. After 28 days of twice per week doxorubicin administration of 6 mg/kg, systemic toxicity (cardiac hypertrophy and weight and hair loss) was not detected with the copper-doxorubicin liposomes but was substantial with ammonium sulfate-loaded doxorubicin liposomes. We then incorporated two strategies designed to enhance efficacy, mTOR inhibition (rapamycin) to slow proliferation and therapeutic ultrasound to enhance accumulation and local diffusion. Tumor accumulation was ~10% ID/g and was enhanced approximately two-fold with the addition of therapeutic ultrasound. After the 28-day course of therapy, syngeneic tumors regressed to a pre-malignant phenotype of ~ (1 mm)3 or could not be detected.

Neuroendocrine (NE) phenotype, seen in >30% of prostate adenocarcinomas (PCa), and NE prostate tumors are implicated in aggressive prostate cancer. Formation of NE prostate tumors in the TRAMP mouse model was suppressed in mice lacking the ubiquitin ligase Siah2, which regulates HIF-1alpha availability. Cooperation between HIF-1alpha and FoxA2, a transcription factor expressed in NE tissue, promotes recruitment of p300 to transactivate select HIF-regulated genes, Hes6, Sox9, and Jmjd1a. These HIF-regulated genes are highly expressed in metastatic PCa and required for hypoxia-mediated NE phenotype, metastasis in PCa, and the formation of NE tumors. Tissue-specific expression of FoxA2 combined with Siah2-dependent HIF-1alpha availability enables a transcriptional program required for NE prostate tumor development and NE phenotype in PCa.

The v-Crk protein was originally isolated as the oncogene fusion product of the CT10 chicken retrovirus. Cellular homologues of v-Crk include Crk, which encodes two alternatively spliced proteins (CrkI and CrkII), and CrkL. Though CrkI/II proteins are elevated in several types of cancer, including breast, the question of whether these Crk adaptor proteins can promote breast cancer has not been addressed. We created a transgenic mouse model that allows the expression of CrkII through the hormonally responsive mouse mammary tumor virus promoter. During puberty, transgenic mice were found to have delayed ductal outgrowth, characterized by increased collagen surrounding the terminal end buds. In post-pubertal mice, precocious ductal branching was observed and associated with increased proliferation. Focal mammary tumors appeared in a subset of animals, with a latency of approximately 15 months. Mouse mammary tumor virus/CrkII tumors showed high levels of Crk protein as well as various cytokeratin markers characteristic of their respective tumor pathologies. This study demonstrates that the precise expression of CrkII is critical for integrating signals for ductal outgrowth and branching morphogenesis during mammary gland development. Furthermore, this study provides evidence for a potential role of CrkII in integrating signals for breast cancer progression in vivo, which has important implications for elevated CrkII observed in human cancer.

To provide a continuous and prolonged delivery of the substrate D-luciferin for bioluminescence imaging in vivo, luciferin was encapsulated into liposomes using either the pH gradient or acetate gradient method. Under optimum loading conditions, 0.17 mg luciferin was loaded per mg of lipid with 90-95% encapsulation efficiency, where active loading was 6 to 18-fold higher than that obtained with passive loading. Liposomal luciferin in a long-circulating formulation had good shelf stability, with 10% release over 3-month storage at 4 degrees C. Pharmacokinetic profiles of free and liposomal luciferin were then evaluated in transgenic mice expressing luciferase. In contrast to rapid in vivo clearance of free luciferin (t(1/2)=3.54 min), luciferin encapsulated into long-circulating liposomes showed a prolonged release over 24h. The first-order release rate constant of luciferin from long-circulating liposomes, as estimated from the best fit of the analytical model to the experimental data, was 0.01 h(-1). Insonation of luciferin-loaded temperature-sensitive liposomes directly injected into one tumor of Met1-luc tumor-bearing mice resulted in immediate emission of light. Systemic injection of luciferin-loaded long-circulating liposomes into Met1-luc tumor-bearing mice, followed by unilateral ultrasound-induced hyperthermia, produced a gradual increase in radiance over time, reaching a peak at 4-7 h post-ultrasound.

Background Results from previous studies have suggested that breast cancer risk correlates with total lifetime exposure to estrogens and that early-life 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure or diets high in fat can also increase cancer risk. Objectives Because both TCDD and diet affect the estrogen pathway, we examined how TCDD and a high-fat diet (HFD) interact to alter breast cancer susceptibility. Methods We exposed pregnant female FVB/NJ mice (12.5 days postcoitus) to 1 μg/kg TCDD or vehicle; at parturition, the dams were randomly assigned to a low-fat diet (LFD) or a high-fat diet (HFD). Female offspring were maintained on the same diets after weaning and were exposed to 7,12-dimethylbenz[a]anthracene on postnatal days (PNDs) 35, 49, and 63 to initiate mammary tumors. A second cohort of females was treated identically until PND35 or PND49, when mammary gland morphology was examined, or PND50, when mammary gland mRNA was analyzed. Results We found that maternal TCDD exposure doubled mammary tumor incidence only in mice fed the HFD. Among HFD-fed mice, maternal TCDD exposure caused rapid mammary development with increased Cyp1b1 (cytochrome P450 1B1) expression and decreased Comt (catechol-O-methyltransferase) expression in mammary tissue. Maternal TCDD exposure also increased mammary tumor Cyp1b1 expression. Conclusions Our data suggest that the HFD increases sensitivity to maternal TCDD exposure, resulting in increased breast cancer incidence, by changing metabolism capability. These results provide a mechanism to explain epidemiological data linking early-life TCDD exposure and diets high in fat to increased risk for breast cancer in humans.

Epithelial–mesenchymal transition tumorigenesis in the mouse has been described for over 100 years using various terms and with little comprehension of the underlying mechanisms. Recently, epithelial–mesenchymal transition tumors have been recognized in mammary glands of genetically engineered mice. This review provides a historical perspective and the current observations in the context of some of the key molecular biology. The biology of mouse mammary epithelial–mesenchymal transition tumorigenesis is discussed with comparisons to human breast cancer.

Clinical studies have revealed that social support improves the outcome of cancer patients, whereas epidemiologic studies suggest that social isolation increases the risk of death associated with several chronic diseases. However, the precise molecular consequences of an unfavorable social environment have not been defined. To do so, robust, reproducible preclinical models are needed to study the mechanisms whereby an adverse environment affects gene expression and cancer biology. Because random assignment of inbred laboratory mice to well-defined social environments allows accurate and repeated measurements of behavioral and endocrine parameters, transgenic mice provide a preclinical framework with which to begin to determine gene-environment mechanisms. In this study, we found that female C3(1)/SV40 T-antigen mice deprived of social interaction from weaning exhibited increased expression of genes encoding key metabolic pathway enzymes in the premalignant mammary gland. Chronic social isolation was associated with up-regulated lipid synthesis and glycolytic pathway gene expression—both pathways are known to contribute to increased breast cancer growth. Consistent with the expression of metabolic genes in premalignant mammary tissue, isolated mice subsequently developed a significantly larger mammary gland tumors burden compared with group-housed mice. Endocrine evaluation confirmed that isolated mice developed a heightened corticosterone stress response compared with group-housed mice. Together, these transdisciplinary studies show for the first time that an adverse social environment is associated with altered mammary gland gene expression and tumor growth. Moreover, the identification of specific alterations in metabolic pathways gene expression favoring tumor growth suggests potential molecular biomarkers and/or targets (e.g., fatty acid synthesis) for preventive intervention in breast cancer.

We previously identified a SNF1/AMPK-related protein kinase, Hunk, from a mammary tumor arising in an MMTV-neu transgenic mouse. The function of this kinase is unknown. Using targeted deletion in mice, we now demonstrate that Hunk is required for the metastasis of c-myc-induced mammary tumors, but is dispensable for normal development. Reconstitution experiments revealed that Hunk is sufficient to restore the metastatic potential of Hunk-deficient tumor cells, as well as defects in migration and invasion, and does so in a manner that requires its kinase activity. Consistent with a role for this kinase in the progression of human cancers, the human homologue of Hunk is overexpressed in aggressive subsets of carcinomas of the ovary, colon, and breast. In addition, a murine gene expression signature that distinguishes Hunk-wild type from Hunk-deficient mammary tumors predicts clinical outcome in women with breast cancer in a manner consistent with the pro-metastatic function of Hunk in mice. These findings identify a direct role for Hunk kinase activity in metastasis and establish an in vivo function for this kinase.

Overexpression and/or amplification of the ErbB-2 oncogene as well as inactivation of the PTEN tumor suppressor are two important genetic events in human breast carcinogenesis. To address the biological impact of conditional inactivation of PTEN on ErbB-2-induced mammary tumorigenesis, we generated a novel transgenic mouse model that utilizes the murine mammary tumor virus (MMTV) promoter to directly couple expression of activated ErbB-2 and Cre recombinase to the same mammary epithelial cell (MMTV-NIC). Disruption of PTEN in the mammary epithelium of the MMTV-NIC model system dramatically accelerated the formation of multifocal and highly metastatic mammary tumors, which exhibited homogenous pathology. PTEN-deficient/NIC-induced tumorigenesis was associated with an increase in angiogenesis. Moreover, inactivation of PTEN in the MMTV-NIC mouse model resulted in hyperactivation of the phosphatidylinositol 3′-kinase/Akt signaling pathway. However, like the parental strain, tumors obtained from PTEN-deficient/NIC mice displayed histopathological and molecular features of the luminal subtype of primary human breast cancer. Taken together, our findings provide important implications in understanding the molecular determinants of mammary tumorigenesis driven by PTEN deficiency and ErbB-2 activation and could provide a valuable tool for testing the efficacy of therapeutic strategies that target these critical signaling pathways.

Prolactin-secreting pituitary adenomas are common spontaneous lesions in aging FVB females. Prolactin-secreting pituitary proliferations play a significant role in mouse mammary tumorigenesis generally producing adenosquamous carcinomas. Since genetically engineered FVB mice are frequently used to study mammary tumor biology, we have examined a cohort of 64 aging wild-type FVB/N females to establish the prevalence and the nature of spontaneous mammary and pituitary tumors. Tissues from mammary and pituitary glands were studied by histopathology and immunohistochemistry. Of the 64 examined mice, 20 had pituitary tumors and 20 had mammary tumors. Mammary and pituitary tumors were associated in 17 mice. All pituitary tumors were prolactin-positive by immunohistochemistry and classified as prolactinomas. Fourteen mammary tumors, including 12 cases with and 2 without concurrent prolactinomas, were adenocarcinomas with different combinations of epithelial growth patterns. Five mice with prolactinomas had mammary tumors characterized by the epithelial-mesenchymal transition (EMT) phenotype. Estrogen receptor alpha (ERα)-positivity was observed for 14 of the 18 mammary tumors tested, including both adenocarcinomas with nuclear immunoreactivity and EMT-phenotype tumors with both nuclear and cytoplasmic immunoreactivity. No immunoreactivity for the progesterone receptor was observed. This study confirms that spontaneous prolactinomas and mammary tumors are both common and significantly associated lesions in FVB mice. Parity and age represented risk factors for the development of these tumors. Compared with previous reports, prolactinoma-associated mammary tumors displayed a broader morphologic spectrum, including cases with the EMT phenotype. The elevated number of prolactinoma-associated and ERα-positive mammary tumors opens intriguing possibilities concerning the role of ERα cytoplasmic localization during EMT tumorigenesis.

Tumor suppressor gene PTEN is important in the initiation and progression of human prostate carcinoma, whereas the role of TP53 remains controversial. Since Pten/Trp53 double conditional knockout mice show earlier onset and fast progression of prostate cancer when compared to Pten knockout mice, we asked whether heterozygosity of these two tumor suppressor genes was sufficient to accelerate prostatic tumorigenesis. To answer this question we examined prostatic lesion progression of Pten/Trp53 double heterozygous mice and a series of controls such as Pten heterozygous, Pten conditional knockout, Trp53 heterozygous and Trp53 knockout mice. Tissue recombination of adult prostatic epithelium coupled with embryonic rat seminal vesicle mesenchyme was used as a tool to stimulate prostatic epithelial proliferation. In our study, high-grade prostatic intraepithelial neoplasia (PIN) was found with high frequency at 8 weeks post-tissue recombination transplantation. PIN lesions in Pten/Trp53 double heterozygous mice were more severe than those seen in Pten heterozygous alone. Furthermore, morphologic features attributable to Pten or Trp53 loss appeared to be enhanced in double heterozygous tissues. LOH analysis of Pten and Trp53 in genomic DNA collected from high-grade PIN lesions in Pten heterozygous and Pten/Trp53 double heterozygous mice showed an intact wild-type allele for both genes in all samples examined. In conclusion, simultaneous heterozygosity of Pten and Trp53 accelerates prostatic tumorigenesis in this mouse model of prostate cancer independently of loss of heterozygosity of either gene.

To explore the in vivo significance of ShcA during mammary tumorigenesis, we used mice expressing several phosphotyrosine-deficient ShcA alleles under the control of their endogenous promoter. We show that all three ShcA tyrosine phosphorylation sites are involved in the early stages of mammary tumour progression, including loss of the myoepithelial cell layer surrounding hyperplasias and during progression to carcinoma. We have determined that signals emanating from Y313 are important for tumour cell survival, whereas Y239/240 transduce signals promoting tumour vascularization. We further demonstrate that loss of ShcA expression in mammary epithelial cells abrogates tumour development. This study is the first to directly demonstrate that signalling downstream from the ShcA adaptor protein is critical for breast cancer development.

ErbB-2 overexpression and amplification occurs in 15% to 30% of human invasive breast carcinomas associated with poor clinical prognosis. Previously, we have shown that four ErbB-2/Neu tyrosine-autophosphorylation sites within the cytoplasmic tail of the receptor recruit distinct adaptor proteins and are sufficient to mediate transforming signals in vitro. Two of these sites, representing the growth factor receptor binding protein 2 (Grb2; Neu-YB) and the Src homology and collagen (Shc; Neu-YD) binding sites, can induce mammary tumorigenesis and metastasis. Here, we show that transgenic mice bearing the two other ErbB-2 autophosphorylation sites (Neu-YC and Neu-YE) develop metastatic mammary tumors. A detailed comparison of biological profiles among all Neu mutant mouse models revealed that Neu-YC, Neu-YD, and Neu-YE mammary tumors shared similar pathologic and transcriptional features. By contrast, the Neu-YB mouse model displayed a unique pathology with a high metastatic potential that correlates with a distinct transcriptional profile, including genes that promote malignant tumor progression such as metalloproteinases and chemokines. Furthermore, Neu-YB tumor epithelial cells showed abundant intracellular protein level of the chemokine CXCL12/SDF-1α, which may reflect the aggressive nature of this Neu mutant mouse model. Taken together, these findings indicate that activation of distinct Neu-coupled signaling pathways has an important impact on the biological behavior of Neu-induced tumors. [Cancer Res 2007;67(16):7579–88]

Advanced cervical cancer remains a vexing clinical challenge despite screening programs. Many of these cancers are hypoxic, and expression of the alpha subunit of the major regulator of the hypoxic cellular response, the transcription factor hypoxia-inducible factor-1 (HIF-1), is correlated with poor prognosis. Here, we tested a functional role for HIF-1alpha in pathogenesis of cervical cancer in estrogen-treated transgenic mice. Double-transgenic (DTG) mice developed locally invasive cervical cancers 70 times larger than K14-HPV16 mice. In vivo bromodeoxyuridine incorporation was elevated in DTG cancers without a significant increase in apoptosis. HIF-1alpha gain of function did not up-regulate canonical HIF-1 targets in premalignant DTG cervices, in contrast to elevation of these targets in K14-HIF-1alpha transgenic cervices. The DTG transcriptional signature included up-regulation of mRNAs encoding cytokines and chemokines, immune signaling molecules, extracellular proteases, and cell motility factors, as well as reduced expression of cell adhesion and epithelial differentiation genes. Importantly, a set of gene markers derived from the DTG transcriptome predicted cervical cancer progression in patients. This study suggests a novel paradigm for HIF-1 function evident in multistage carcinogenesis as opposed to established malignancies, including interaction with viral oncogenes to induce multiple genomic networks in premalignancy that fosters the development of advanced cervical cancer.

One-third of patients with breast cancer overexpress the ERBB2 receptor tyrosine kinase, which is associated not only with a more aggressive phenotype but also reduced responsiveness to hormonal therapies. Over the past two decades, many ERBB2 mouse models for breast cancer have conclusively shown that this receptor has a causal role in breast cancer development. These mouse models have also enabled the mechanisms controlling tumour growth, angiogenesis, metastasis, dormancy and recurrence in ERBB2-positive breast cancer to be elucidated. In addition, a mouse model has recently been described that accurately recapitulates many of the hallmarks associated with the early stages of the human disease.

Introduction The diversity of human breast cancer subtypes has led to the hypothesis that breast cancer is actually a number of different diseases arising from cells at various stages of differentiation. The elusive nature of the cell(s) of origin thus hampers approaches to eradicate the disease. Methods Clonal cell lines were isolated from primary transgenic polyomavirus middle T (PyVmT) luminal tumors. Mammary cancer stem cell (MaCSC) properties were examined by immunofluorescence, flow cytometry, differentiation assays and in vivo tumorigenesis. Results Clonal cell lines isolated from primary PyVmT mouse mammary luminal tumors can differentiate into luminal, myoepithelial, alveolar and adipocyte lineages. Upon orthotopic injection, progeny of a single cell follow a pattern of progression from ductal carcinoma in situ, to adenoma, adenocarcinoma and epithelial metastasis that recapitulates the transgenic model. Tumors can evolve in vivo from hormone receptor-positive to hormone receptor-negative Her2-positive, or triple negative CD44hi basal-like and claudin-low tumors. Contrary to the current paradigm, we have defined a model in which multiple tumor subtypes can originate from a single multipotent cancer stem cell that undergoes genetic and/or epigenetic evolution during tumor progression. As in human tumors, the more aggressive tumor subtypes express nuclear p53. Tumor cell lines can also be derived from these more advanced tumor subtypes. Conclusions Since the majority of human tumors are of the luminal subtype, understanding the cell of origin of these tumors and how they relate to other tumor subtypes will impact cancer therapy. Analysis of clonal cell lines derived from different tumor subtypes suggests a developmental hierarchy of MaCSCs, which may provide insights into the progression of human breast cancer. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0615-y) contains supplementary material, which is available to authorized users.

Epithelial Mesenchymal Transition (EMT) is the transdifferentiation of epithelial cells into a mesenchymal phenotype. This process occurs during embryogenesis but also in wound healing and in tumors. The neoplastic EMT is characterized by variably complete shedding of epithelial architectural features and acquisition of mesenchymal traits. In immunohistochemistry a variable coexpression of cytokeratins, vimentin or alpha-smooth muscle actin with loss of E-cadherin and other interepithelial adhesion molecules is characteristic. Such transition is associated with mutations both at the genetic (somatic) and epigenetic levels and is believed to confer a more advantageous phenotype for local and distant spread of cancer cells. Mammary carcinoma can exhibit EMT features in humans and mice and it tends to occur more frequently in women with tumors bearing a worse prognosis such as the claudin low subtype within the triple negative cancer. Missense mutation of TP53 is one of the most common mutations in cancer and it is frequently found in EMT tumor types, often with a more aggressive behavior. The current literature and survey of our mouse EMT cases in the Genomic Pathology Center image archives demonstrate a synergy between p53 and EMT that is independent of the initiating oncogene. However, p53 mutation is not sufficient or causal for EMT. Moreover, despite the local malignant behavior, processes such as spontaneous metastases and Mesenchymal Epithelial Transition (MET) appear not to be as frequent and obvious as previously hypothesized.

Hormone receptor status is an integral component of decision-making in breast cancer management. IHC4 score is an algorithm that combines hormone receptor, HER2, and Ki-67 status to provide a semiquantitative prognostic score for breast cancer. High accuracy and low interobserver variance are important to ensure the score is accurately calculated; however, few previous efforts have been made to measure or decrease interobserver variance. We developed a Web-based training tool, called "Score the Core" (STC) using tissue microarrays to train pathologists to visually score estrogen receptor (using the 300-point H score), progesterone receptor (percent positive), and Ki-67 (percent positive). STC used a reference score calculated from a reproducible manual counting method. Pathologists in the Athena Breast Health Network and pathology residents at associated institutions completed the exercise. By using STC, pathologists improved their estrogen receptor H score and progesterone receptor and Ki-67 proportion assessment and demonstrated a good correlation between pathologist and reference scores. In addition, we collected information about pathologist performance that allowed us to compare individual pathologists and measures of agreement. Pathologists' assessment of the proportion of positive cells was closer to the reference than their assessment of the relative intensity of positive cells. Careful training and assessment should be used to ensure the accuracy of breast biomarkers. This is particularly important as breast cancer diagnostics become increasingly quantitative and reproducible. Our training tool is a novel approach for pathologist training that can serve as an important component of ongoing quality assessment and can improve the accuracy of breast cancer prognostic biomarkers.

We present a status report from the NCI Mouse Models of Human Cancers Consortium (MMHCC) Precancers Workshop held November 8 and 9, 2004. An expert panel, the Mouse Models Group (MMG) evaluated the status of mouse models of precancer emphasizing genetically engineered mouse models, especially of lining epithelium and their utilitarian value to human carcinogenesis. An outline of the background for the panel’s considerations is provided with examples of past and current precancerous lesions in mice. The experimental use of oncogenic viruses and chemical carcinogens in mice led to operational definitions of initiation, promotion, and preneoplasia Preneoplastic and precancerous lesions are found in these models. In this precancer concept, most preneoplastic lesions are considered as potentially precancerous or at least an earlier stage in cancer development than typical pre-invasive epithelial lesions, which are often seen in these mouse models. Genetically engineered mice, used to test the oncogenicity of individual genes, develop precancers that are initiated by defined molecular and histopathologic changes. The mouse can be used to isolate and study precancers in detail, thereby providing a level of biological understanding not readily available in clinical disease. These studies suggest that genetically engineered mice are very useful preclinical models for chemoprevention and therapy.

2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) has been implicated as a major mutagenic heterocyclicamine in the human diet and is carcinogenic in the rat prostate. To validate PhIP-induced rat prostatic neoplasia as a model of human prostate cancer progression, we sought to study the earliest histologic and morphologic changes in the prostate and to follow progressive changes over time. We fed sixty-seven 5-week-old male Fischer F344 rats with PhIP (400 ppm) or control diets for 20 weeks, and then sacrificed animals for histomorphologic examination at the ages of 25, 45, and 65 weeks. Animals treated with PhIP showed significantly more inflammation (P = .002, > .001, and .016 for 25, 45, and 65 weeks, respectively) and atrophy (P = .003, > .001, and .006 for 25, 45, and 65 weeks, respectively) in their prostate glands relative to controls. Prostatic intraepithelial neoplasia (PIN) occurred only in PhIP-treated rats. PIN lesions arose in areas of glandular atrophy, most often in the ventral prostate. Atypical cells in areas of atrophy show loss of glutathione S-transferase π immunostaining preceding the development of PIN.None of the animals in this study developed invasive carcinomas, differing from those in previous reports. Overall, these findings suggest that the pathogenesis of prostatic neoplasia in the PhIP-treated rat prostate proceeds from inflammation to postinflammatory proliferative atrophy to PIN.

The aryl hydrocarbon receptor (AhR) is a transcription factor that mediates the inhibitory effects of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) on prostate growth and also modulates normal prostate development. This is evidenced by AhR null mice (Ahr � /� ) having smaller dorsolateral and anterior prostates, even though all prostate lobes remain histologically normal. To test the hypothesis that loss of the AhR increases the rate of prostate carcinogenesis, the incidence of macroscopic prostate tumors was determined in Ahr +/+ , Ahr +/� and Ahr � /� C57BL/6J transgenic adenocarcinoma of the mouse prostate (TRAMP) mice at 35, 70, 105, 140, 175 and 210 days of age. From 140 days, prostate tumor incidence was greater in Ahr � /� (60%) and Ahr +/� (43%) mice than in Ahr +/+ mice (16%). Allele quantification did not indicate a loss of the wild-type Ahr allele in heterozygous TRAMP tumors, suggesting that tumor formation in these mice was not due to a loss of Ahr heterozygosity. Prostatic SV40 large T antigen mRNA expression and protein localization were comparable in TRAMP mice of each Ahr genotype. Prostates from all mice of each Ahr genotype were histologically indistinguishable, exhibiting diffuse epithelial hyperplasia by 105 days of age. mRNA expression and protein localization for molecular markers of neuroendocrine differentiation, including chromogranin A and neuropilin-1, were elevated in prostate tumors compared to tumor-free ventral prostates, regardless of Ahr genotype or age. Taken together, these results demonstrate that the Ahr inhibits prostate carcinogenesis in C57BL/6J TRAMP mice by interfering with neuroendocrine differentiation.

Purpose: Rapamycin has been shown to have antitumor effects in various tumor models. To study the effect of rapamycin at different stages of breast cancer development, we used two unique mouse models of breast cancer with activated phosphatidylinositol 3-kinase (PI3K) pathway. Met-1 tumors are highly invasive and metastatic, and mammary intraepithelial neoplasia-outgrowths (MIN-O), a model for human ductal carcinoma in situ, are transplantable premalignant mammary lesions that develop invasive carcinoma with predictable latencies. Both of these models were derived from mammary lesions in Tg(MMTV-PyV-mT) mice. Experimental Design: Met-1 tumors were used to study the effect of rapamycin treatment on invasive disease. Transplanted MIN-O model was used to study the effect of rapamycin on premalignant mammary lesions. Animals were in vivo micro–positron emission tomography imaged to follow the lesion growth and transformation to tumor during the treatment. Cell proliferation, angiogenesis, and apoptosis was assayed by immunohistochemistry. Results: Rapamycin inhibited in vitro tumor cell proliferation and in vivo Met-1 tumor growth. The growth inhibition was correlated with dephosphorylation of mammalian target of rapamycin (mTOR) targets. Rapamycin treatment significantly reduced the growth of the premalignant MIN-O lesion, as well as tumor incidence and tumor burden. Growth inhibition was associated with reduced cell proliferation and angiogenesis and increased apoptosis. Conclusions: In PyV-mT mouse mammary models, rapamycin inhibits the growth of premalignant lesions and invasive tumors. Although the inhibitory effect of rapamycin was striking, rapamycin treatment did not completely obliterate the lesions.

To determine how different epithelial cell types respond to the same oncogenic stimulation, we have used a modified human keratin 18 gene to conditionally express the polyomavirus middle T antigen (PyMT) oncogene in simple epithelial tissues of transgenic mice. Activation of PyMT expression by transgenic Cre recombinase in mammary epithelial cells resulted in carcinomas in all bitransgenic females. PyMT expression induced by K18-driven Cre in internal epithelial organs resulted in pancreatic acinar metaplasia and ductal dysplasia with remarkable desmoplastic stromal responses in all 25 bitransgenic mice. Hepatoma formation with altered lipid metabolism and gastric adenocarcinoma occurred in 96 and 54% of these mice, respectively. Elevated PyMT RNA expression also correlated with intraepithelial neoplasia in the prostate. Activated Erk2 was found in mammary tumors, pancreatic tissues, and affected livers. Hes1 RNA, a target of Notch signaling that has been implicated downstream of Ras pathway activation, was elevated in pancreatic and liver lesions. The variety of responses of different epithelia to PyMT demonstrates the importance of the differentiated state in interpreting oncogenic signals.

Because each change in the evolution of a cancer is predicated on the effects of previous events, a full understanding of selective changes and their effect on tumor progression can only be understood in the context of appropriate initiating events. Here, we define the effect of pRb function inactivation in prostate epithelium on both the initiation of prostate cancer and the establishment of selective pressures that lead to diminished Pten function and tumor evolution. Using genetically engineered mice, we show that inactivation of the pRb family proteins (Rb/p107/p130) induces epithelial proliferation and apoptosis and is sufficient to produce prostatic intraepithelial neoplasia (PIN) lesions. Over time, adenocarcinomas develop in all mice with no evidence of neuroendocrine tumors. Apoptosis is dependent on Pten function and not p53, unlike other epithelial cell types tested previously. Consequently, Pten hemizygosity reduces apoptosis by 50%, accelerating progression to adenocarcinomas with heterogeneous composition. Heterogeneity is associated with concurrent Pten haploinsufficiency and focal selective progression to complete Pten loss, which yields distinct tumor properties. Given that this analysis models the apparent timing of highly penetrant events in human prostate cancer, observed effects may recapitulate the natural evolution of prostate cancer development.

Two cell lines, Met-1fvb2 and DB-7fvb2, with different metastatic potential, were derived from mammary carcinomas in FVB/N-Tg(MMTV-PyVmT) and FVB/N-Tg(MMTV-PyVmT Y315F/Y322F ) mice, transplanted into syngeneic FVB/N hosts and characterized. The lines maintain a stable morphological and biological phenotype after multiple rounds of in vitro culture and in vivo transplantation. The Met-1fvb2 line derived from a FVB/N-Tg(MMTV-PyVmT) tumor exhibits invasive growth and 100% metastases when transplanted into the females FVB/N mammary fat pad. The DB-7fvb2 line derived from the FVB/N-Tg(MMTV-PyVmT Y315F/Y322F ) with a “double base” modification at Y315F/Y322F exhibits more rapid growth when transplanted into the mammary fat pad, but a lower rate of metastasis (17%). The Met1fvb2 cells show high activation of AKT, while DB-7fvb2 cells show very low levels of AKT activation. The DNA content and gene expression levels of both cell lines are stable over multiple generations. Therefore, these two cell lines provide a stable, reproducible resource for the study of metastasis modulators, AKT molecular pathway interactions, and gene target and marker discovery.

Rapid advances in generating new mouse genetic models for lung neoplasia provide a continuous challenge for pathologists and investigators. Frequently, phenotypes of new models either have no precedents or are arbitrarily attributed according to incongruent human and mouse classifications. Thus, comparative characterization and validation of novel models can be difficult. To address these issues, a series of discussions was initiated by a panel of human, veterinary, and experimental pathologists during the Mouse Models of Human Cancers Consortium (NIH/National Cancer Institute) workshop on mouse models of lung cancer held in Boston on June 20–22, 2001. The panel performed a comparative evaluation of 78 cases of mouse and human lung proliferative lesions, and recommended development of a new practical classification scheme that would (a) allow easier comparison between human and mouse lung neoplasms, (b) accommodate newly emerging mouse neoplasms, and (c) address the interpretation of benign and preinvasive lesions of the mouse lung. Subsequent discussions with additional experts in pulmonary pathology resulted in the current proposal of a new classification. It is anticipated that this classification, as well as the complementary digital atlas of virtual histological slides, will help investigators and pathologists in their characterization of new mouse models, as well as stimulate further research aimed at a better understanding of proliferative lesions of the lung.

Modern pathologists have been challenged to “validate” mouse models of human cancer. Validation requires matching of morphological attributes of the model to human disease. Computers can assist in the validation process. However, adequate controlled, computer-readable vocabularies that can match terms do not currently exist in mouse pathology. Further, current standard diagnostic terminologies do not include the new concepts discussed here such as pathway pathology and mammary intraepithelial neoplasia. The terminologies must be revised and improved to meet the challenge. Human medicine has traditionally used “guilt-by-association” to validate interpretations of disease. Experimental pathology uses experimental verification exemplified by “test-by-transplantation.” Genetically Engineered Mice (GEM) develop unique tumor phenotypes bringing new structural-functional insights and reevaluation of concepts. Novel GEM-related tumors appear in all organ systems but mouse models of human breast cancer are prototypes. For example, mammary tumors induced by Mouse Mammary Tumor Virus (MMTV), chemical, radiation or other carcinogenic stimuli have limited phenotypes. These “spontaneous” or induced mammary tumors have never resembled human breast cancers. GEM tumors created with genes associated with human cancer are strikingly different. GEM tumors have unique histological phenotypes. Depending on the genes, the tumors may: 1) resemble MMTV-induced tumors, 2) display “signature” phenotypes, and 3) mimic human breast cancers. The phenotypes can be placed into structural and functional clusters with shared characteristics leading to the concepts of Pathway Pathology: tumor phenotype reflects the genotype.

Nitric oxide (NO) and its metabolites are implicated in carcinogenesis and metastasis. Both stimulatory and inhibitory effects of NO have been reported in relation to breast cancer and its role in the development of malignancies and metastasis remains uncertain. We have used the polyomavirus middle T antigen (PyV-mT) targeted to the mouse mammary gland and bred into an inducible NO synthase (iNOS)deficient C57Bl/6 strain to examine a role for nitric oxide in modulating tumors that develop in the complex environment of the whole animal. The development of hyperplasias was delayed to the extent that the earliest palpable tumors arose 2– 4 weeks later in PyV-mT/iNOS / mice compared with PyV-mT/iNOS / mice, identifying a role for iNOS in early events in mammary tumor formation. Tumors that did develop in PyV-mT/iNOS / mice were characteristically well differentiated and had a cribriform pattern. Other tumors were myoepithelial adenocarcinomas with uniform nuclear size. In contrast, mice capable of iNOS activity typically developed solid nodular adenocarcinomas with a high mitotic index and pleomorphic nuclei. No significant effect of iNOS deficiency was found on vascular density in hyperplasias or tumors by examining CD31-positive vessels. The infiltration of lesions by macrophages, cells capable of significant NO production, remained unchanged in PyV-mT/iNOS / mice. Metastatic potential was retained by PyV-mT-transformed epithelium in the absence of iNOS, indicating that NO production by iNOS is not essential for this process. These results indicate a role for iNOS in tumorigenesis, particularly in the regulation of early events. © 2003 Wiley-Liss, Inc.