Mammary Oncogenesis Research Articles

Common Spontaneous Tumors and Tumor-like Lesions in 70 Pet Rodents and Negative MMTV Detection in Mammary Tumors.

Compared to the number of studies on the neoplasms of laboratory rodents, fewer studies have focused on spontaneous neoplasms in pet rodents. Notably, the mouse mammary tumor virus (MMTV) is associated with mammary tumors in rodents. In this study, 77 tumors and tumor-like lesions of biopsy samples were collected from 70 pet rodents, including hamsters (n = 47), guinea pigs (n = 16), unknown species (n = 4), rats (n = 2), and a gerbil. Fifty tumors were collected from 47 hamsters, in which the most common tumors were mammary tumors (13/50), followed by fibrosarcoma (9/50), mast cell tumors (4/50), and squamous cell carcinoma (4/50). The collected subtypes of mammary tumors in hamsters included tubular carcinoma (n = 5), tubular adenoma (n = 4), carcinoma and malignant myoepithelioma (n = 1), simple tubular carcinoma (n = 1), adenosquamous carcinoma (n = 1), and tubulopapillary adenoma (n = 1). In addition, twenty tumors were collected from guinea pigs, in which the most common tumor was lipoma (6/20), followed by adenocarcinoma of the mammary gland (4/20), trichofolliculoma (2/20), and collagenous hamartomas (2/20). In guinea pigs, the subtypes of mammary gland tumors were tubular carcinoma (n = 2), tubular and solid carcinoma (n = 1), and tubulopapillary carcinoma (n = 1). In 20 cases of mammary tumors, MMTV was not detected, implicating no evidence of MMTV infection in mammary oncogenesis in pet rodents in Taiwan.

Abstract 6605: Understanding the role of sumoylated Etv1 in mammary oncogenesis

Abstract The Small Ubiquitin-like Modifier (SUMO) pathway is a major post-translational regulatory mechanism involved in a variety of cellular functions including the DNA damage response, transcriptional regulation, and carcinogenesis. Recent discoveries by our group demonstrated that inhibiting the SUMO pathway can repress the growth of breast cancer xenografts and Neu-driven mammary oncogenesis in a genetically engineered mouse model with conditional knockout (CKO) of the SUMOylation E2 conjugating enzyme, Ube2i. However, despite the consistency of the findings, there remains considerable debate concerning the mechanisms through which SUMO inhibition represses tumorigenesis. Our downstream analysis suggested ETS Translocation Variant 1, encoded by ETV1 gene, acts as a SUMO-sensitive transcription factor that drives mammary oncogenesis and cancer growth. To examine oncogenic properties of SUMO-conjugated ETV1 in breast cancer, we cloned HA-tagged WT Etv1, SUMO-resistant Etv15xKR, or SUMO mimetic SUMO-1-Etv15xKR into a lentiviral vector with a GFP cassette. Proliferation, colony formation in soft agar, and in vivo xenograft assays were evaluated in the Ube2iF/F cell line derived from a tumor in a MMTV-c-neu/Ube2ifl/fl animal. We did ChIP-seq in Ube2iF/F cells using SUMO-1 and Etv1 antibodies to examine the effects of SUMO conjugation on Etv1 genomic occupancy. RNA-seq using siEtv1 or AdCre was also performed to compare changes in gene expression with SUMO-conjugated Etv1 genomic occupancy. Inhibition of SUMOylation via AdCre caused a significant reduction in proliferation and colony formation in vitro and completely block the formation of flank xenografts in vivo. Interestingly, expressing a SUMO-resistant ETV15xKR rescued in vitro growth effects, whereas a SUMO mimetic SUMO-1-ETV15xKR had no effect on restoring proliferation. On the contrary, in vivo xenograft assays demonstrated that the Etv15xKR-SUMO1 efficiently induced tumor formation despite the loss of Ube2i while the sumo-resistant Etv15xKR was unable to rescue tumorigenesis. Bulk RNA-seq and ChIP seq performed demonstrated different patterns of gene regulation between sumoylated and unsumoylated Etv1. Gene set enrichment analysis (GSEA) showed genes associated with cancer stemness were significantly enriched in cells expressing Etv15xKR-SUMO1. Collectively, these observations support Etv1 acting as a SUMO-sensitive transcription factor that drives a stem cell phenotype necessary for mammary oncogenesis and progression. Citation Format: Zhijie Li, Kelsey E. Koch, Dakota T. Thompson, Dana M. Van der Heide, Jeremy Chang, Christopher M. Franke, Mohammed O. Suraju, Anna C. Beck, Allison W. Lorenzen, Jeffrey R. White, Mikhail V. Kulak, David K. Meyerholz, Colin Kenny, Ronald J. Weigel. Understanding the role of sumoylated Etv1 in mammary oncogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6605.

Nano Diosgenin Abates DMBA Induced Renal and Hepatic Toxicities: Biochemical and Histopathological Evaluation on the Breast Cancer Model

Background: : Nature-based pharmaceuticals are now becoming an integral aspect of toxic-free healthcare therapies. Diosgenin (DN), a unique phyto steroidal sapogenin, seems to be explicitly employed as a core ingredient in countless traditional and patented Chinese medicines owing to its epic multilayered therapeutic treasure. Objective: The prime intent of the current study was to probe the hepato- and nephro- ameliorating impact of Diosgenin encapsulated chitosan nanoparticles (DN@CS-NPS) on 7,12-dimethylbenz(a)anthracene (DMBA) mediated rat mammary oncogenesis. Methods: A single dosage of DMBA (25 mg/kg body weight) was injected to induce breast cancer. Oral administration of DN (10 mg/kg body weight) and DN@CS-NPS (5 mg/kg body weight) was used to medicate DMBA administeredtumor-bearing rats just after the emergence of a tumor. Following the experimental duration, biochemical and histopathological (H&E) analyses have been carried out. Results: Here, we noticed that there is an escalated level of liver and kidney biomarkers, phase-I detoxification enzymes, lipid peroxidative marker, total cholesterol (TC), phospholipids (PL), triglycerides (TG), and free fatty acids (FFA), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), total lipase (TL) as well as diminished levels of phase – II detoxification enzymes, enzymatic and non-enzymatic antioxidants, high-density lipoprotein (HDL), lipoprotein lipase (LPL) and lecithin acyltransferase (LCAT) in the plasma, liver and kidney tissues of DMBA-induced rats with renal and hepatic histopathological alterations. Conversely,oral treatment of DN@CS-NPS substantially reduced their tiers to near-normal levels. Conclusion: Thus, our observations suggested that DN@CS-NP is an impactful hepato- and nephro- therapeutic agent that might have a significant influence on breast cancer overfree DN.

Breast Cancer and Prolactin - New Mechanisms and Models.

The pathogenesis of breast cancer is driven by multiple hormones and growth factors. One of these, prolactin (PRL), contributes to both mammary differentiation and oncogenesis, and yet the basis for these disparate effects has remained unclear. The focus of this review is to examine and place into context 2 recent studies that have provided insight into the roles of PRL receptors and PRL in tumorigenesis and tumor progression. One study provides novel evidence for opposing actions of PRL in the breast being mediated in part by differential PRL receptor (PRLr) isoform utilization. Briefly, homomeric complexes of the long isoform of the PRLr (PRLrL-PRLrL) promotes mammary differentiation, while heteromeric complexes of the intermediate and long PRLr (PRLrI-PRLrL) isoforms trigger mammary oncogenesis. Another study describes an immunodeficient, prolactin-humanized mouse model, NSG-Pro, that facilitates growth of PRL receptor-expressing patient-derived breast cancer xenografts. Evidence obtained with this model supports the interactions of physiological levels of PRL with estrogen and ERBB2 gene networks, the modulatory effects of PRL on drug responsiveness, and the pro-metastatic effects of PRL on breast cancer. This recent progress provides novel concepts, mechanisms and experimental models expected to renew interest in harnessing/exploiting PRLr signaling for therapeutic effects in breast cancer.

FHER2, PR, ER, Ki-67 and Cytokeratin 5/6 Expression in Benign Feline Mammary Lesions.

Simple SummaryFeline mammary neoplasias are highly prevalent in domestic cats and present many similarities to their human counterparts. Since information about benign feline mammary lesions is still scarce and often controversial, studies using a wider panel of oncological biomarkers are necessary to understand their potential contribution to malignant lesions. This study analyzed 47 benign lesions from 27 queens, regarding the expression of estrogen and progesterone receptors (ER and PR, respectively), fHER2 protein and two malignancy indicators (Ki-67 and CK 5/6). Our results showed that most of the lesions were ER positive (91.5%), PR negative (63.8%), fHER2 negative (64.4%), Ck 5/6 negative (76.6%) and with a low Ki-67 index (78.7%). Additionally, significant correlations were found between younger ages and ER positivity and between larger lesions and negative PR status. Our results highlight the importance of estrogen receptors in the development of benign feline mammary lesions, further contributing to the development of preventive and monitoring strategies in feline mammary oncology.Biomarkers are essential in the characterization of neoplastic lesions and aid not only in the classification of the nature of the lesions, but also in the understanding of their ontogeny, development and prognosis. In cats, while mammary carcinomas are increasingly being characterized, information on their benign lesions is still scarce. Indeed, a better characterization of benign lesions could have an important role in unravelling mammary oncogenesis, similar to that in human breast cancer. Thus, in this study, the expression of five markers was analyzed in 47 benign mammary lesions (hyperplasia, dysplasia and benign tumors) collected from 27 queens. Dysplastic and hyperplastic lesions were the most common (41/47, 81.7%). Most of the lesions were classified as ER positive (43/47, 91.5%), PR negative (30/47, 63.8%), fHER2 negative (29/47, 64.4%), CK 5/6 negative (36/47, 76.6%) and with a low Ki-67 index (37/47, 78.7%). Statistical analysis revealed a correlation between younger ages and ER positivity (p = 0.013) and between larger lesions and negative PR status (p = 0.038). These results reinforce the importance of evaluating the expression of the ER status, prevalent in benign lesions, as a putative precursor in cancer progression.

Activation Versus Inhibition of IGF1R: A Dual Role in Breast Tumorigenesis.

Historically, the body of literature surrounding the insulin-like growth factor type 1 receptor (IGF1R) has described a largely pro-tumorigenic role in breast cancer cells and in several transgenic or xenograft mouse models of breast cancer. Interestingly, however, more recent evidence has emerged that suggests an additional, previously undescribed, tumor and metastasis suppressive function for IGF1R in both human breast tumors and mammary oncogenesis in mice. These seemingly conflicting reports can be reconciled when considering what is currently known about IGF1R function in the context of tissue development and cancer as it relates to cellular growth, proliferation, and differentiation. In this mini review, we will summarize the currently existing data with a particular focus on mouse models that have been developed to study IGF1R function in mammary development, tumorigenesis, and metastasis in vivo and propose hypotheses for how both the tumor-promoting and tumor-suppressing schools of thought regarding IGF1R in these histological contexts are compatible.

Parity-induced changes to mammary epithelial cells control NKT cell expansion and mammary oncogenesis.

SUMMARYPregnancy reprograms mammary epithelial cells (MECs) to control their responses to pregnancy hormone re-exposure and carcinoma progression. However, the influence of pregnancy on the mammary microenvironment is less clear. Here, we used single-cell RNA sequencing to profile the composition of epithelial and non-epithelial cells in mammary tissue from nulliparous and parous female mice. Our analysis indicates an expansion of γδ natural killer T-like immune cells (NKTs) following pregnancy and upregulation of immune signaling molecules in post-pregnancy MECs. We show that expansion of NKTs following pregnancy is due to elevated expression of the antigen-presenting molecule CD1d on MECs. Loss of CD1d expression on post-pregnancy MECs, or overall lack of activated NKTs, results in mammary oncogenesis. Collectively, our findings illustrate how pregnancy-induced changes modulate the communication between MECs and the immune microenvironment and establish a causal link between pregnancy, the immune microenvironment, and mammary oncogenesis.

Impact of Stilbenes as Epigenetic Modulators of Breast Cancer Risk and Associated Biomarkers.

With the recent advancement of genetic screening for testing susceptibility to mammary oncogenesis in women, the relevance of the gene−environment interaction has become progressively apparent in the context of aberrant gene expressions. Fetal exposure to external stressors, hormones, and nutrients, along with the inherited genome, impact its traits, including cancer susceptibility. Currently, there is increasing interest in the role of epigenetic biomarkers such as genomic methylation signatures, plasma microRNAs, and alterations in cell-signaling pathways in the diagnosis and primary prevention of breast cancer, as well as its prognosis. Polyphenols like natural stilbenes have been shown to be effective in chemoprevention by exerting cytotoxic effects that can stall cell proliferation. Besides possessing antioxidant properties against the DNA-damaging effects of reactive oxygen species, stilbenes have also been observed to modulate cell-signaling pathways. With the increasing trend of early-life screening for hereditary breast cancer risks, the potency of different phytochemicals in harnessing the epigenetic biomarkers of breast cancer risk demand more investigation. This review will explore means of exploiting the abilities of stilbenes in altering the underlying factors that influence breast cancer risk, as well as the appearance of associated biomarkers.

Serine residues 726 and 780 have nonredundant roles regulating STAT5a activity in luminal breast cancer

In breast cancer, prolactin-induced activation of the transcription factor STAT5a results from the phosphorylation of STAT5a tyrosine residue 694. However, its role in mammary oncogenesis remains an unsettled debate as STAT5a exhibits functional dichotomy with both pro-differentiative and pro-proliferative target genes. Phosphorylation of STAT5a serine residues, S726 and S780, may regulate STAT5a in such a way to underlie this duality. Given hematopoiesis studies showing phospho-serine STAT5a as necessary for transformation, we hypothesized that serine phosphorylation regulates STAT5a activity to contribute to its role in mammary oncogenesis, specifically in luminal breast cancer. Here, phosphorylation of S726-, S780-, and Y694-STAT5a in response to prolactin in MCF7 luminal breast cancer cells was investigated with STAT5a knockdown and rescue with Y694F-, S726A-, or S780A-STAT5a, where the phospho-sites were mutated. RNA-sequencing and subsequent Ingenuity Pathway Analysis predicted that loss of each phospho-site differentially affected both prolactin-induced gene expression as well as functional pathways of breast cancer (e.g. cell survival, proliferation, and colony formation). In vitro studies of anchorage-independent growth and proliferation confirmed distinct phenotypes: whereas S780A-STAT5a decreased clonogenicity, S726A-STAT5a decreased proliferation in response to prolactin compared to wild type STAT5a. Collectively, these studies provide novel insights into STAT5a activation in breast cancer pathogenesis.

Investigating the pathophysiological role of RANKL in mammary gland density and oncogenesis in osteoporotic TgRANKL mice

Investigating the pathophysiological role of RANKL in mammary gland density and oncogenesis in osteoporotic TgRANKL mice

Lysophosphatidic acid-RAGE axis promotes lung and mammary oncogenesis via protein kinase B and regulating tumor microenvironment

BackgroundReceptor for advanced glycation end products (RAGE) is a multi-ligand transmembrane receptor of the immunoglobulin superfamily. Lysophosphatidic acid (LPA) is a ligand for RAGE and is involved in physiological and pathophysiological conditions including cancer. However, RAGE-LPA axis is unexplored in lung and mammary cancer.MethodsRAGE was silenced in A549, MDA MB-231 and MCF7 using RAGE shRNA. For in vitro tumorigenesis, we performed wound healing, colony formation, cell proliferation and invasion assays. Evaluation of expression of oncogenes, EMT markers and downstream signaling molecules was done by using western blot and immunohistochemistry. For subcellular expression of RAGE, immunofluorescence was done. In vivo tumorigenesis was assessed by intraperitoneal injection of cancer cells in nude mice.ResultsHere we show RAGE mediated profound increase in proliferation, migration and invasion of lung and mammary cancer cells via LPA in Protein kinase B (PKB) dependent manner. LPA mediated EMT transition is regulated by RAGE. In vivo xenograft results show significance of RAGE in LPA mediated lung and mammary tumor progression, angiogenesis and immune cell infiltration to tumor microenvironment.ConclusionOur results establish the significance and involvement of RAGE in LPA mediated lung and mammary tumor progression and EMT transition via RAGE. RAGE-LPA axis may be a therapeutic target in lung and mammary cancer treatment strategies.EdVB1f1yrz4Do4i3nWt_5KVideo

Regulation of breast cancer oncogenesis by the cell of origin's differentiation state.

Human breast cancer which affects 1/8 women is rare at a cellular level. Even in the setting of germline BRCA1/BRCA2, which is present in all breast cells, solitary cancers or cancers arising at only several foci occur. The overwhelming majority of breast cells (109–1012 cells) resist transformation. Our hypothesis to explain this rareness of transformation is that mammary oncogenesis is regulated by the cell of origin’s critical window of differentiation so that target cells outside of this window cannot transform. Our novel hypothesis differs from both the multi-hit theory of carcinogenesis and the stem/progenitor cell compartmental theory of tumorigenesis and utilizes two well established murine transgenic models of breast oncogenesis, the FVB/N-Tg (MMTV-PyVT)634Mul/J and the FVB-Tg (MMTV-ErbB2) NK1Mul/J. Tail vein fibroblasts from each of these transgenics were used to generate iPSCs. When select clones were injected into cleared mammary fat pads, but not into non-orthotopic sites of background mice, they exhibited mammary ontogenesis and oncogenesis with the expression of their respective transgenes. iPSC clones, when differentiated along different non-mammary lineages in vitro, were also not able to exhibit either mammary ontogenesis or oncogenesis in vivo. Therefore, in vitro and in vivo regulation of differentiation is an important determinant of breast cancer oncogenesis.

SAT-139 STAT5A Regulation by Serine Phosphorylation in Breast Cancer

The neuroendocrine hormone prolactin (PRL) and its cognate receptor (PRLr) have been implicated in the pathogenesis of breast cancer. PRL signaling relies on activating kinases such as the tyrosine kinase Jak2 and serine/threonine kinases ERK1/2, Nek3, PI3K, and AKT. In the canonical pathway of PRL signaling, Jak2 phosphorylates the transcription factor Stat5a at tyrosine residue 694 (pY694-Stat5a), preceding Stat5a nuclear translocation and transcriptional activity. However, Stat5a exists with functional duality as a transcription factor, having both pro-differentiative and pro-proliferative target genes. Other Stat family members (Stats 1, 3, and 6) have been shown to have transcriptional activity in the un-phosphorylated (upY) state, distinct from that of pY-Stat activity. This distinction (upY vs. pY) may underlie the duality of Stat5a, coupled with additional regulatory non-canonical post-translational modifications. Within this notion, Stat5a contains two serine residues, S726 and S780, whose phosphorylation are necessary for hematopoietic transformation. However, their functions in PRL-mediated breast cancer pathogenesis have not been examined. We hypothesize that Stat5a serine phosphorylation regulates Stat5a nuclear activity in a non-canonical fashion, contributing to its role in mammary oncogenesis. As shown in a tissue microarray (TMA), human breast cancer tissues express both pS726- and pS780-Stat5a. Nuclear Allred score for pS726-Stat5a increases two-fold with increasing tumor grade, with no difference in staining associated with estrogen or progesterone receptor (ER, PR) status, nor other clinical characteristics. Likewise, patient derived xenograft (PDX) tumors of various molecular subtypes express pS726- and pS780-Stat5a. Phosphorylation of S726-Stat5a is PRL-responsive in vitro. Pharmacologic inhibition of ERK1/2 prevents this phosphorylation, uncovering a novel pathway in which ERK1/2 mediates Stat5a activity in response to PRL in breast cancer. To examine the functional significance of Stat5a serine phosphorylation in vitro, we have performed Stat5a knockdown (KD) in the breast cancer cell line MCF7. Following Stat5a KD, cells were rescued with phospho-site specific Stat5a mutant constructs. Characteristics of breast cancer examined in these mutation-carrying cells, including anchorage-independent growth and proliferation, show distinct phenotypes compared to controls. PRL-induced expression of the CISH gene is significantly decreased up to 65% in the mutation-carrying cells compared to wild type Stat5a. Mechanistic studies will examine the ability of these Stat5a mutants to undergo nuclear translocation and interact with other transcription factors. Collectively, these studies have the potential to provide novel insights into the role of the non-canonical pathway of Stat5a activation in breast cancer pathogenesis.

Abstract BL1: Deciphering stem and progenitor cells to understand breast cancer

Abstract Breast cancer is a highly heterogeneous disease at the molecular and pathological levels. To understand heterogeneity and potential ‘cells of origin’ of breast cancer, it is important to dissect the normal mammary epithelial differentiation hierarchy as well as the cellular composition of breast tumors. Discrete populations of mouse and human mammary epithelial cells have been isolated on the basis of cell surface marker expression, yielding three primary populations including a stem cell-enriched population and two luminal populations. Their responsiveness to female steroid hormones was investigated, revealing the importance of paracrine regulation of ER/PR– stem/progenitor cells by ER/PR+ luminal ‘hormone sensor’ cells. RANKL was found to be a key paracrine effector of progesterone signaling, underscoring the central role that female hormones play in normal mammary gland homeostasis and cancer. Consistent with this observation, a RANK+ luminal progenitor cell was found to be the likely target cell that gives rise to breast cancer in BRCA1 mutation carriers. Pre-clinical studies further indicate that it may be possible to target this ‘culprit’ cell with the RANKL inhibitor denosumab. This finding is now being tested in a randomized phase III prevention study, ‘BRCA-P’. In further studies, a quiescent stem cell that remains hormone responsive has been identified. Lineage tracing, combined with high resolution 3D imaging (visualizing large regions of intact tissue), was carried out to track stem and progenitor cells in situ. These studies have revealed the presence of long-lived stem cells that replenish mammary epithelium, while unipotent progenitors appear to play an essential role in sustaining the independent lineages on a day-to-day basis. In order to generate a comprehensive single cell expression resource and explore molecular heterogeneity in the mouse mammary gland, single cell profiling at key developmental stages was performed. This has revealed novel intermediates and unexpected changes in the transcriptional landscape that could inform a role for distinct cell types during mammary oncogenesis. To gain insight into cancer heterogeneity and clonal dynamics at cellular resolution, 3D imaging was combined with lineage tracing to track specific cells during cancer development. Through the application of a novel pipeline, the epithelial-to-mesenchymal transition (EMT) was found to be a frequent event, thus highlighting the inherent plasticity of mammary tumors. These findings are currently being extended to human tumors to further understand inter- and intra-tumoral heterogeneity. In parallel, our group has established a bank of patient-derived xenograft (PDX) and patient derived organoid (PDO) models to facilitate the transfer of laboratory findings to the clinic. These serve as useful pre-clinical models to test promising agents. Initial efforts have focused on BH3 mimetics, a new class of drug that targets the BCL-2 family of pro-survival proteins. This work is leading to early phase clinical trials, currently focused on patients with metastatic ER+ breast cancer. Citation Format: JE Visvader, GJ Lindeman. Deciphering stem and progenitor cells to understand breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr BL1.

Functional Evaluation of ZNF350 Missense Genetic Variants Associated with Breast Cancer Susceptibility.

ZNF350, a BRCA1-interacting protein, could mediate BRCA1-induced sequence-specific transcriptional repression of several genes, including GADD45α. As a potential breast cancer susceptibility gene, single nucleotide polymorphisms (SNPs), especially missense SNPs, may influence the transcriptional repression of its target tumor suppressor genes and individuals' breast cancer risk. Using the gene-based haplotype-tagging SNPs strategy, we evaluated the association between six ZNF350 polymorphisms and breast cancer risk in a case-control set from a northern Chinese population. The impact of ZNF350 variations on transcriptional repression of GADD45α was also examined. It was found that ZNF350 rs2278420 (L66P) and rs2278415 (S501R) missense genetic variants are in complete linkage disequilibrium and have a significant impact on inter-individual susceptibility to breast cancer. Additionally, ZNF350 GGCGT or GGCGC haplotype is also associated with a significantly increased breast cancer risk compared with the GGCAC haplotype. ZNF350 L66P variant modifies the risk of breast cancer not only by itself but also in a gene-environment interaction manner with age, age at menarche, menopause status, or estrogen receptor status. Interestingly, we observed that ZNF350 L66P and S501R SNPs could weaken the capability of ZNF350-mediated GADD45α transcription repression and it may be an underlying mechanism of the observed epidemiological associations. Our results highlight ZNF350 as an important gene in human mammary oncogenesis and ZNF350 missense genetic polymorphisms confer susceptibility to breast cancer.

Abstract P6-05-04: mRNA expression of PTK6V1 and PTKV2 in human breast cancer

Abstract BACKGROUND: Protein tyrosine kinase 6 (PTK6) is an intracellular protein that is upregulated in several human cancers and its localization to the plasma-membrane facilitates its various oncogenic roles in breast cancer cell proliferation, survival, and migration. The full length protein (PTK6V1) and alternative splice variant (PTK6V2) seem to play different roles with the latter being negative regulator of the former. In this study, the level of mRNA expression of PTK6V1 and PTK6V2 were assessed in normal and malignant breast tissue using real time Q-PCR in a cohort of women with breast cancer and correlated to conventional clinico-pathological parameters and clinical outcome. MATERIALS AND METHODS: Breast cancer tissues (n = 127) and normal background tissues (n = 33) were collected immediately after excision during surgery. Following RNA extraction, reverse transcription was carried out and transcript levels were determined using real-time quantitative PCR and normalized against beta-actin expression. Transcript levels within the breast cancer specimens were compared to the normal background tissues and analysed against TNM stage, nodal involvement, tumour grade and clinical outcome over a 10 year follow-up period. RESULTS: The median copy number of transcripts of PTK6V1 were higher in malignant compared with normal breast tissue (23 vs.7) and overall increased with advancing tumour stage (374 vs. 13 for TNM3 vs. TNM1 respectively p=0.019 and 374 vs.23 for TNM3 vs. TNM2 p=0.0244). however these associations did reach statistical significance. PTK6V1 levels were associated with oestrogen receptor (ER) positivity (p = 0.061). The transcript levels were significantly higher in patients who developed recurrence (p=0.03) or died of breast cancer (p=0.003). PTK6V2 transcript levels were generally higher in normal breast tissue than in malignant tissues and decreased with increasing tumour stage and grade however these associations did not reach stastical significance. After a median follow up of 10 years, there was a trend for higher PTK6V2 expression to be associated with longer overall survival (OS) and disease free survival (DFS). PTK6V2/PTK6V1 ratio was a significant predictor of OS. Conclusions Our observations suggest that the two variants of PTK6 play opposing roles in mammary oncogenesis. These findings could have prognostic and therapeutic implications. Citation Format: El Hage Chehade H, Harvey A, Wazir U, Jiang WG, Mokbel K. mRNA expression of PTK6V1 and PTKV2 in human breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-05-04.

Immune Checkpoint Inhibitors in Triple Negative Breast Cancer: What is the Evidence?

Breast cancer is the most prevalent breast cancer in women. Despite our deeper understanding of mammary oncogenesis, 20% of breast cancer patients develop metastasis and die. There is a dire need for novel therapies especially for more aggressive subtypes like triple negative (TNBC) and HER2 positive breast cancers. For long, breast cancer was thought to be less immunogenic in nature compared to other solid malignancies like melanoma and non-small cell lung cancer, both later cancers yielding great results with immunotherapy. As we learn more about the role of tumor microenvironment in tumor occurrence, progression and response to therapy, there is growing evidence that TNBC is more immunogenic in nature compared to other breast cancers, which encouraged researchers to investigate its response to immune check point inhibitors. TNBC is characterized by a high level of immune cell infiltration including tumor infiltrating lymphocytes (TILs) which have been associated with better outcomes. The prognostic values of TILs and PD-L1 expression on tumor cells have been an area of interest and remain uncertain to date. The hope is that future trials will unravel more answers about the complex interaction between TILs and tumor cells in TNBC and help develop biomarkers to identify responders to checkpoint inhibitors. In this review, we discuss the immunogenic nature of TNBC, the clinical relevance of TILs and PD-L1 expression and the promising results of early phase trials of checkpoint blockade.

Lack of placental transfer of certolizumab pegol during pregnancy: Results from crib, a prospective, postmarketing, multicenter, pharmacokinetic study

Although substantial evidence has demonstrated that parity and 17β-estradiol (E2) reduce mammary carcinogenesis, it is not clear how this protection is conferred. Thus, we examined the effects of parity and E2 treatment in the mammary glands of ovariectomized 15 week-old virgin mice, 15 week-old primiparous mice, and 9 month-old retired breeders. E2 treatment significantly increased lipid peroxidation, protein carbonylation, and protein nitrosylation in the virgin mice, but not in the age-matched primiparous mice or retired breeders. Mammary gland expression of the oxidative stress response protein Cu/Zn superoxide dismutase was consistently reduced in all of the E2-treated mice regardless of parity. Expression of the oxidative stress and DNA repair protein apurinic endonuclease (Ape1) was significantly increased only in the mammary glands of the E2-treated retired breeders. These findings suggest that E2 and parity help to reduce mammary oncogenesis by maintaining the structure and function of proteins, lipids, and DNA.

Immunosurveillance and immunoediting in MMTV-PyMT-induced mammary oncogenesis

ABSTRACTEvidence of cancer immunosurveillance and immunoediting processes has been primarily demonstrated in mouse models of chemically induced oncogenesis. Although these models are very tractable, they are characterized by high mutational loads that represent a minority of human cancers. In this study, we sought to determine whether cancer immunosurveillance and immunoediting could be demonstrated in a more clinically relevant oncogene-induced model of carcinogenesis, the MMTV-PyMT (PyMT) mammary carcinoma model. This model system in the FVB/NJ strain background was previously used to demonstrate that adaptive immunity had no role in limiting primary cancer formation and in fact promoted metastasis, thus calling into question whether cancer immunosurveillance operated in preventing the development of breast cancer. Our current study in the C57BL/6 strain backgrounds provides a different conclusion, as we report here the existence of an adaptive immunosurveillance of PyMT mammary carcinomas using two independent models of immune deficiency. PyMT mice bred onto a Rag1−/− background or immune suppressed by chronic tacrolimus therapy both demonstrated accelerated development of mammary carcinomas. By generating a bank of cell lines from these animals, we further show that a subset of PyMT cell lines had delayed growth after transplantation into wild-type (WT) syngeneic, but not immune-deficient hosts. This reduced growth rate in immunocompetent animals was characterized by an increase in immune cell infiltration and tissue differentiation. Furthermore, loss of the immune cell infiltration that characterized immunoediting of slow growing cell lines, changed them into fast growing variants capable of progressing in the immunocompetent model. In conclusion, our study provides evidence that immunosurveillance and immunoediting of PyMT-derived cell lines modulate tumor progression in this oncogene-induced model of cancer.

Fatty acid synthase is a primary target of MiR-15a and MiR-16-1 in breast cancer.

Fatty acid synthase (FASN) is upregulated in breast cancer and correlates with poor prognosis. FASN contributes to mammary oncogenesis and serves as a bona fide target in cancer therapies. MicroRNAs inhibit gene expression through blocking mRNA translation or promoting mRNA degradation by targeting their 3′-UTRs. We identified four microRNAs in two microRNA clusters miR-15a-16-1 and miR-497-195 that share a common seed sequence to target the 3′-UTR of the FASN mRNA. In reporter assays, both of these microRNA clusters inhibited the expression of a reporter construct containing the FASN 3′-UTR. However, only ectopic miR-15a-16-1, but not miR-497-195, markedly reduced the levels of endogenous FASN in breast cancer cells. Both miR-15a and miR-16-1 contributes to inhibiting FASN expression and breast cancer cell proliferation. Consistently, a sponge construct consisting of eight repeats of the FASN 3′-UTR region targeted by these microRNAs could markedly increase endogenous FASN levels in mammary cells. When FASN expression was restored by ectopic expression in breast cancer cells, retarded cell proliferation caused by miR-15a-16-1 was partially rescued. In conclusion, we demonstrated that FASN expression is primarily downregulated by miR-15a and miR-16-1 in mammary cells and FASN is one of the major targets of these two tumor suppressive microRNAs.