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Supplemental Table 3 from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Supplementary Figure 2 from Distinct ErbB-2–Coupled Signaling Pathways Promote Mammary Tumors with Unique Pathologic and Transcriptional Profiles

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PDF file - 99K, Expression of SCRIB mRNA in human breast cancer.

Supplementary Table 1 from Tuberous Sclerosis Complex 1: An Epithelial Tumor Suppressor Essential to Prevent Spontaneous Prostate Cancer in Aged Mice

Supplementary Notes from Classification of Proliferative Pulmonary Lesions of the Mouse

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Supplementary Figure 1 from SLITs Suppress Tumor Growth In vivo by Silencing Sdf1/Cxcr4 within Breast Epithelium

Supplementary Figure 4 from Distinct ErbB-2–Coupled Signaling Pathways Promote Mammary Tumors with Unique Pathologic and Transcriptional Profiles

PDF file - 36K, Pathway enrichment analysis for differentially expressed genes between Nkx3.1CE2/+; Ptenf/f; BrafCA/+ and Nkx3.1CE2/+; Ptenf/f mouse prostate tumors

XLS file, 239K, Differentially expressed gene set data for the intact mice.

Supplemental Table 1 from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Animal models, particularly mouse models, play a central role in the study of the etiology, prevention, and treatment of human prostate cancer. While tissue culture models are extremely useful in understanding the biology of prostate cancer, they cannot recapitulate the complex cellular interactions within the tumor microenvironment that play a key role in cancer initiation and progression. The National Cancer Institute (NCI) Mouse Models of Human Cancers Consortium convened a group of human and veterinary pathologists to review the current animal models of prostate cancer and make recommendations about the pathologic analysis of these models. More than 40 different models with 439 samples were reviewed, including genetically engineered mouse models, xenograft, rat, and canine models. Numerous relevant models have been developed over the past 15 years, and each approach has strengths and weaknesses. Analysis of multiple genetically engineered models has shown that reactive stroma formation is present in all the models developing invasive carcinomas. In addition, numerous models with multiple genetic alterations display aggressive phenotypes characterized by sarcomatoid carcinomas and metastases, which is presumably a histologic manifestation of epithelial–mesenchymal transition. The significant progress in development of improved models of prostate cancer has already accelerated our understanding of the complex biology of prostate cancer and promises to enhance development of new approaches to prevention, detection, and treatment of this common malignancy. Cancer Res; 73(9); 2718–36. ©2013 AACR.

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]

PDF file - 204K, Characterization of hSCRIBP305L tumors.

Supplementary Figure 7 from Phosphatase and Tensin Homologue Deleted on Chromosome 10 Deficiency Accelerates Tumor Induction in a Mouse Model of ErbB-2 Mammary Tumorigenesis

Supplementary Figures 1-11, Tables 1-4 from ANCCA/ATAD2 Overexpression Identifies Breast Cancer Patients with Poor Prognosis, Acting to Drive Proliferation and Survival of Triple-Negative Cells through Control of B-Myb and EZH2

Supplemental Fig 6 from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Introduction: Immune checkpoint blockade (ICB) has clinical activity in triple negative breast cancer (TNBC) but is less effective in the ER+HER2- signature, where there is a cold immune microenvironment (IM) and regulatory T cells (Tregs) may suppress effector T cells. Agents that activate the IM by turning cold tumors hot may support ICB. The biguanides hexyl-benzyl-biguanide (HBB) and its bioisostere hexyl-(cuban-1-yl-methyl)-biguanide (HCB) are candidate agents to activate the IM because they potently inhibit biosynthesis of immunosuppressive epoxyeicosatrienoic acids (EETs) and EET-driven oxidative phosphorylation (OXPHOS), while blocking N-glycosylation of immune checkpoint (IC) proteins. We hypothesized that reversal of hypoxia by biguanides in the ovarian dependent ER+HER2- STAT1 KO SSM2ucd mouse mammary carcinoma (MC) model would suppress Tregs and promote effector T cells in the tumor IM. While the SSM2ucd model did not express immune checkpoint protein PD-L1 (B7-H1), it did express related IC proteins B7-H3 and B7-H4. We hypothesized that by inhibiting OXPHOS and reducing N-glycosylation of immune checkpoint proteins, HBB and HCB may promote efficacy of ICB. We chose the SSM2ucd model to test impact of HCB on the ER+ MC IM. Results: SSM2ucd cells exhibited longer tumor latency (60 days) than the basal 4T1 (10 days) and 67NR (20 days) mouse MC models. SSM2ucd tumor reimplantation shortened latency by more than half, to 20 days. Immunohistochemistry showed that B7-H3 and B7-H4 protein levels were 1.2 (P=0.001) and 1.3-fold (P=0.04) higher in reimplanted tumors vs. control. In SSM2ucd cells, HCB inhibited N-glycosylation of B7-H3 (P=0.01) by 35% and B7-H4 (P=0.02) by 45% and suppressed TGFβ induction of B7-H3 by 21% (P=0.02) and B7-H4 by 79% (P=0.001) at 24 hours, while 14,15-EET promoted N-glycosylation of B7-H3 (1.2-fold; P=0.03) and B7-H4 (1.3-fold; P=0.04) at 4 hours. Effects of HBB and HCB on anti-CD3 and anti-CD28 stimulated mouse splenocytes were assayed. The proliferative effects of HBB on CD4+ and CD8+ cells peaked at 12 uM (p Conclusion: B7-H3 and B7-H4 expression inversely correlated with latency of ER+ MC and may represent targets for immune checkpoint antibodies and their drug conjugates. HCB, an inhibitor of OXPHOS and EET biosynthesis, reduced intratumoral hypoxia, increased CD8+ TIL and reduced the Treg:CD8+ ratio, potentially supporting ICB therapy of ER+ MC by turning cold tumors hot. Supported by CDMRP BCRP Grant BC180596, Award Number W81XWH-19-1-0099 Citation Format: Zhijun Guo, Jianxun Lei, Hrishi Venkatesh, David Owen, Adam Bass, Christine Cannon, Joshua McCarra, Brenda Koniar, Craig Flory, Beverly Norris, Robert J. Schumacher, Swaathi Jayaraman, John Hawse, Emmanuel S. Antonarakis, Emanuel F. Petricoin, Julia Wulfkuhle, Robert D. Cardiff, Elizabeth A. Ambrose, Gunda I. Georg, Kaylee L. Schwertfeger, Michael A. Farrar, Brad St. Croix, Matthew P. Goetz, David A. Potter. Hexyl-(cuban-1-yl-methyl)-biguanide (HCB) suppresses N-glycosylation of immune checkpoint proteins B7-H3 and B7-H4, reverses tumor hypoxia, decreases intratumoral regulatory T cells, and increases intratumoral CD8+ T cells in the ovarian dependent ER+HER2- SSM2ucd mammary cancer allograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB102.

Ecdysoneless (ECD) protein is essential for embryogenesis, cell-cycle progression, and cellular stress mitigation with an emerging role in mRNA biogenesis. We have previously shown that ECD protein as well as its mRNA are overexpressed in breast cancer and ECD overexpression predicts shorter survival in patients with breast cancer. However, the genetic evidence for an oncogenic role of ECD has not been established. Here, we generated transgenic mice with mammary epithelium-targeted overexpression of an inducible human ECD transgene (ECDTg). Significantly, ECDTg mice develop mammary hyperplasia, preneoplastic lesions, and heterogeneous tumors with occasional lung metastasis. ECDTg tumors exhibit epithelial to mesenchymal transition and cancer stem cell characteristics. Organoid cultures of ECDTg tumors showed ECD dependency for in vitro oncogenic phenotype and in vivo growth when implanted in mice. RNA sequencing (RNA-seq) analysis of ECDTg tumors showed a c-MYC signature, and alterations in ECD levels regulated c-MYC mRNA and protein levels as well as glucose metabolism. ECD knockdown-induced decrease in glucose uptake was rescued by overexpression of mouse ECD as well as c-MYC. Publicly available expression data analyses showed a significant correlation of ECD and c-MYC overexpression in breast cancer, and ECD and c-MYC coexpression exhibits worse survival in patients with breast cancer. Taken together, we establish a novel role of overexpressed ECD as an oncogenesis driver in the mouse mammary gland through upregulation of c-MYC–mediated glucose metabolism. Implications: We demonstrate ECD overexpression in the mammary gland of mice led to the development of a tumor progression model through upregulation of c-MYC signaling and glucose metabolism.

p110α is a catalytic subunit of phosphoinositide 3-kinase (PI3K), a major downstream effector of receptor tyrosine kinase ErbB2, that is amplified and overexpressed in 20-30% of breast cancers, 40% of which have an activating mutation in p110α. Despite the high frequency of PIK3CA gain-of-function mutations, their prognostic value is controversial. Here, we employ a knock-in transgenic strategy to restrict the expression of an activated form of ErbB2 and p110α kinase domain mutation (p110α

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.

The staging and local management of breast cancer involves the evaluation of the extent and completeness of excision of both the invasive carcinoma component and also the intraductal component or ductal carcinoma in situ. When both invasive ductal carcinoma and coincident ductal carcinoma in situ are present, assessment of the extent and localization of both components is required for optimal therapeutic planning. We have used a mouse model of breast cancer to evaluate the feasibility of applying molecular imaging to assess the local status of cancers in vivo. Multi-tracer positron emission tomography (PET) and magnetic resonance imaging (MRI) characterize the transition from premalignancy to invasive carcinoma. PET tracers for glucose consumption, membrane synthesis, and neoangiogenesis in combination with a Gaussian mixture model-based analysis reveal image-derived thresholds to separate the different stages within the whole-lesion. Autoradiography, histology, and quantitative image analysis of immunohistochemistry further corroborate our in vivo findings. Finally, clinical data further support our conclusions and demonstrate translational potential. In summary, this preclinical model provides a platform for characterizing multistep tumor progression and provides proof of concept that supports the utilization of advanced protocols for PET/MRI in clinical breast cancer imaging.

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.

Summary: Mechanistic target of rapamycin complex 1 (mTORC1) is a master modulator of cellular growth, and its aberrant regulation is recurrently documented within breast cancer. While the small GTPase Rheb1 is the canonical activator of mTORC1, Rheb1-independent mechanisms of mTORC1 activation have also been reported but have not been fully understood. Employing multiple transgenic mouse models of breast cancer, we report that ablation of Rheb1 significantly impedes mammary tumorigenesis. In the absence of Rheb1, a block in tumor initiation can be overcome by multiple independent mutations in Mtor to allow Rheb1-independent reactivation of mTORC1. We further demonstrate that the mTOR kinase is indispensable for tumor initiation as the genetic ablation of mTOR abolishes mammary tumorigenesis. Collectively, our findings demonstrate that mTORC1 activation is indispensable for mammary tumor initiation and that tumors acquire alternative mechanisms of mTORC1 activation. : While Rheb1-dependent mTORC1 activation is well established within mammalian context, Xiao et al. highlight its contribution in mammary tumorigenesis using breast cancer mouse models. Driven by the evolutionary nature of tumorigenesis, tumors devoid of Rheb1 develop with hyperactivating mTOR mutations to restore mTORC1 activity, thus emphasizing alternative mechanisms of activation. Keywords: Rheb, mTOR, tumorigenesis, breast, cancer, mutation

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.

The need for international collaboration in rodent pathology has evolved since the 1970s and was initially driven by the new field of toxicologic pathology. First initiated by the World Health Organization’s International Agency for Research on Cancer for rodents, it has evolved to include pathology of the major species (rats, mice, guinea pigs, nonhuman primates, pigs, dogs, fish, rabbits) used in medical research, safety assessment, and mouse pathology. The collaborative effort today is driven by the needs of the regulatory agencies in multiple countries, and by needs of research involving genetically engineered animals, for “basic” research and for more translational preclinical models of human disease. These efforts led to the establishment of an international rodent pathology nomenclature program. Since that time, multiple collaborations for standardization of laboratory animal pathology nomenclature and diagnostic criteria have been developed, and just a few are described herein. Recently, approaches to a nomenclature that is amenable to sophisticated computation have been made available and implemented for large-scale programs in functional genomics and aging. Most terminologies continue to evolve as the science of human and veterinary pathology continues to develop, but standardization and successful implementation remain critical for scientific communication now as ever in the history of veterinary nosology.

This chapter reviews the normal anatomy, development, and histology of the rodent and human mammary gland. Mature resting, lactating, and atrophic mammary gland changes are discussed. Differences between rodent and human biologic structure and histologic findings are emphasized.

Advancements in technology and digitization have ushered in novel ways of enhancing tissue-based research via digital microscopy and image analysis. Whole slide imaging scanners enable digitization of histology slides to be stored in virtual slide repositories and to be viewed via computers instead of microscopes. Easier and faster sharing of histologic images for teaching and consultation, improved storage and preservation of quality of stained slides, and annotation of features of interest in the digital slides are just a few of the advantages of this technology. Combined with the development of software for digital image analysis, digital slides further pave the way for the development of tools that extract quantitative data from tissue-based studies. This review introduces digital microscopy and pathology, and addresses technical and scientific considerations in slide scanning, quantitative image analysis, and slide repositories. It also highlights the current state of the technology and factors that need to be taken into account to insure optimal utility, including preanalytical considerations and the importance of involving a pathologist in all major steps along the digital microscopy and pathology workflow.

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.

Tissue localization of immune cells is critical to the study of disease processes in mouse models of human diseases. However, immunohistochemistry (IHC) for immune cell phenotyping in mouse tissue sections presents specific technical challenges. For example, CD4 and CD8 have been difficult to detect using IHC on formalin-fixed and paraffin-embedded mouse tissue, prompting alternative methods. We investigated the use of formalin-free zinc-salt fixation (ZN) and optimized IHC protocols for detecting a panel of immune cell–related markers (CD3, CD4, CD8, Foxp3, B220, F4/80, CD68, and major histocompatibility complex [MHC] class-I, MHC class-II, and Gr-1). The IHC results for these markers were compared on mouse spleen tissue treated with neutral buffered formalin (NBF) or ZN with or ZN without antigen retrieval (AR). Whereas CD4 and CD8 were not detected in NBF-treated tissue, all markers were detected in ZN-treated tissue without AR. Thus, the use of ZN treatment for IHC staining can be a good tool for studying immunoreactive lesions in tissues.

Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1(-/-) mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1(WT) or cyclin D1(KE) in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1(KE) induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1(WT) and cyclin D1(KE) to the genes governing CIN. We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis.

Background Stat1 gene-targeted knockout mice (129S6/SvEvTac-Stat1 tm1Rds) develop estrogen receptor-positive (ER+), luminal-type mammary carcinomas at an advanced age. There is evidence for both host environment as well as tumor cell-intrinsic mechanisms to initiate tumorigenesis in this model. In this report, we summarize details of the systemic and mammary pathology at preneoplastic and tumor-bearing time points. In addition, we investigate tumor progression in the 129:Stat1 −/− host compared with wild-type 129/SvEv, and we describe the immune cell reaction to the tumors. Methods Mice housed and treated according to National Institutes of Health guidelines and Institutional Animal Care and Use Committee-approved methods were evaluated by histopathology, and their tissues were subjected to immunohistochemistry with computer-assisted quantitative image analysis. Tumor cell culture and conditioned media from cell culture were used to perform macrophage (RAW264.7) cell migration assays, including the 129:Stat1 −/−-derived SSM2 cells as well as control Met1 and NDL tumor cells and EpH4 normal cells. Results Tumorigenesis in 129:Stat1 −/− originates from a population of FoxA1+ large oval pale cells that initially appear and accumulate along the mammary ducts in segments or regions of the gland prior to giving rise to mammary intraepithelial neoplasias. Progression to invasive carcinoma is accompanied by a marked local stromal and immune cell response composed predominantly of T cells and macrophages. In conditioned media experiments, cells derived from 129:Stat1 −/− tumors secrete both chemoattractant and chemoinhibitory factors, with greater attraction in the extracellular vesicular fraction and inhibition in the soluble fraction. The result appears to be recruitment of the immune reaction to the periphery of the tumor, with exclusion of immune cell infiltration into the tumor. Conclusions 129:Stat1 −/− is a unique model for studying the critical origins and risk reduction strategies in age-related ER+ breast cancer. In addition, it can be used in preclinical trials of hormonal and targeted therapies as well as immunotherapies. Electronic supplementary material The online version of this article (doi:10.1186/s13058-017-0892-8) contains supplementary material, which is available to authorized users.

Aberrant activation of β-catenin through its activity as a transcription factor has been observed in a large proportion of human malignancies. Despite the improved understanding of the β-catenin signaling pathway over the past three decades, attempts to develop therapies targeting β-catenin remain challenging, and none of these targeted therapies have advanced to the clinic. In this study, we show that part of the challenge in antagonizing β-catenin is caused by its dual functionality as a cell adhesion molecule and a signaling molecule. In a mouse model of basal ErbB2 receptor tyrosine kinase 2 (ErbB2)-positive breast cancer (ErbB2KI), which exhibits aberrant β-catenin nuclear signaling, β-catenin haploinsufficiency induced aggressive tumor formation and metastasis by promoting the disruption of adherens junctions, dedifferentiation, and an epithelial to mesenchymal transition (EMT) transcriptional program. In contrast to the accelerated tumor onset observed in the haploid-insufficient ErbB2 tumors, deletion of both β-catenin alleles in the ErbB2KI model had only a minor impact on tumor onset that further correlated with the retention of normal adherens junctions. We further showed that retention of adherens junctional integrity was caused by the up-regulation of the closely related family member plakoglobin (γ-catenin) that maintained both adherens junctions and the activation of Wnt target genes. In contrast to the ErbB2KI basal tumor model, modulation of β-catenin levels had no appreciable impact on tumor onset in an ErbB2-driven model of luminal breast cancer [murine mammary tumor virus promoter (MMTV-NIC)]. These observations argue that the balance of junctional and nuclear β-catenin activity has a profound impact on tumor progression in this basal model of ErbB2-positive breast cancer.

Scribble (SCRIB) localizes to cell–cell junctions and regulates establishment of epithelial cell polarity. Loss of expression of SCRIB functions as a tumor suppressor in Drosophila and mammals; conversely, overexpression of SCRIB promotes epithelial differentiation in mammals. Here, we report that SCRIB is frequently amplified, mRNA overexpressed, and protein is mislocalized from cell–cell junctions in human breast cancers. High levels of SCRIB mRNA are associated with poor clinical prognosis, identifying an unexpected role for SCRIB in breast cancer. We find that transgenic mice expressing a SCRIB mutant [Pro 305 to Leu (P305L)] that fails to localize to cell–cell junctions, under the control of the mouse mammary tumor virus long terminal repeat promoter, develop multifocal hyperplasia that progresses to highly pleomorphic and poorly differentiated tumors with basal characteristics. SCRIB interacts with phosphatase and tensin homolog (PTEN) and the expression of P305L, but not wild-type SCRIB, promotes an increase in PTEN levels in the cytosol. Overexpression of P305L, but not wild-type SCRIB, activates the Akt/mTOR/S6K signaling pathway. Human breast tumors overexpressing SCRIB have high levels of S6K but do not harbor mutations in PTEN or PIK3CA, identifying SCRIB amplification as a mechanism of activating PI3K signaling in tumors without mutations in PIK3CA or PTEN. Thus, we demonstrate that high levels of mislocalized SCRIB functions as a neomorph to promote mammary tumorigenesis by affecting subcellular localization of PTEN and activating an Akt/mTOR/S6kinase signaling pathway. Cancer Res; 74(11); 3180–94. ©2014 AACR.

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.

Advancements in liposomal drug delivery have produced long circulating and very stable drug formulations. These formulations minimize systemic exposure; however, unfortunately, therapeutic efficacy has remained limited due to the slow diffusion of liposomal particles within the tumor and limited release or uptake of the encapsulated drug. Here, the carboxyl-terminated CRPPR peptide, with affinity for the receptor neuropilin-1 (NRP), which is expressed on both endothelial and cancer cells, was conjugated to liposomes to enhance the tumor accumulation. Using a pH sensitive probe, liposomes were optimized for specific NRP binding and subsequent cellular internalization using in vitro cellular assays. Liposomes conjugated with the carboxyl-terminated CRPPR peptide (termed C-LPP liposomes) bound to the NRP-positive primary prostatic carcinoma cell line (PPC-1) but did not bind to the NRP-negative PC-3 cell line, and binding was observed with liposomal peptide concentrations as low as 0.16mol%. Binding of the C-LPP liposomes was receptor-limited, with saturation observed at high liposome concentrations. The identical peptide sequence bearing an amide terminus did not bind specifically, accumulating only with a high (2.5mol%) peptide concentration and adhering equally to NRP positive and negative cell lines. The binding of C-LPP liposomes conjugated with 0.63mol% of the peptide was 83-fold greater than liposomes conjugated with the amide version of the peptide. Cellular internalization was also enhanced with C-LPP liposomes, with 80% internalized following 3h incubation. Additionally, fluorescence in the blood pool (~40% of the injected dose) was similar for liposomes conjugated with 0.63mol% of carboxyl-terminated peptide and non-targeted liposomes at 24h after injection, indicating stable circulation. Prior to doxorubicin treatment, in vivo tumor accumulation and vascular targeting were increased for peptide-conjugated liposomes compared to non-targeted liposomes based on confocal imaging of a fluorescent cargo, and the availability of the vascular receptor was confirmed with ultrasound molecular imaging. Finally, over a 4-week course of therapy, tumor knockdown resulting from doxorubicin-loaded, C-LPP liposomes was similar to non-targeted liposomes in syngeneic tumor-bearing FVB mice and C-LPP liposomes reduced doxorubicin accumulation in the skin and heart and eliminated skin toxicity. Taken together, our results demonstrate that a carboxyl-terminated RXXR peptide sequence, conjugated to liposomes at a concentration of 0.63mol%, retains long circulation but enhances binding and internalization, and reduces toxicity.

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.