Loss of MXRA8 Delays Mammary Tumor Development and Impairs Metastasis.

Matrix-remodeling-associated protein 8 or MXRA8 is a transmembrane protein that can bind arthritogenic alpha viruses like the Chikungunya virus and provide viral entry into cells. MXRA8 can also interact with integrin ß3 and thus possibly regulate cell-cell interactions and binding to the extracellular matrix. While MXRA8 has been associated with reduced survival in patients with colorectal and renal clear cell cancers, the role of MXRA8 in breast cancer remains largely unexplored. Therefore, the aim of this research was to determine the role of MXRA8 in breast cancer by knocking out MXRA8 in the human triple-negative breast cancer cell line MDA-MB-231. The loss of MXRA8 reduced cell proliferation in vitro but had no effect on apoptosis or migration in cultured cells. However, the loss of MXRA8 significantly delayed tumor development and reduced metastatic dissemination to the lungs in a xenograft model. RNA sequencing identified three genes, ADMATS1, TIE1, and BMP2, whose expression were significantly reduced in MXRA8-knockout tumors compared to control tumors. MXRA8 staining of a human breast cancer tissue array revealed higher levels of MXRA8 in primary tumors and metastases of aggressive tumor subtypes (TNBC and HER2+) compared to less aggressive, ER+ breast cancers. Our findings demonstrate for the first time that MXRA8 regulates the progression of human TNBC possibly through influencing the interaction of tumor cells with their microenvironment.

Distinct Requirements for Adaptor Proteins NCK1 and NCK2 in Mammary Gland Development.

The adaptor proteins NCK1 and NCK2 are well-established signalling nodes that regulate diverse biological processes including cell proliferation and actin dynamics in many tissue types. Here we have investigated the distribution and function of Nck1 and Nck2 in the developing mouse mammary gland. Using publicly available single-cell RNA sequencing data, we uncovered distinct expression profiles between the two paralogs. Nck1 showed widespread expression in luminal, basal, stromal and endothelial cells, while Nck2 was restricted to luminal and basal cells, with prominent enrichment in hormone-sensing luminal subtypes. Next, using mice with global knockout of Nck1 or Nck2, we assessed mammary gland development during and after puberty (5, 8 and 12 weeks of age). Mice lacking Nck1 or Nck2 displayed significant defects in ductal outgrowth and branching at 5 weeks compared to controls, and the defects persisted in Nck2 knockout mice at 8 weeks before normalizing at 12 weeks. These defects were accompanied by an increase in epithelial cell proliferation at 5 weeks and a decrease at 8 weeks in both Nck1 and Nck2 knockout mice. We also profiled expression of several key genes associated with mammary gland development at these timepoints and detected temporal changes in transcript levels of hormone receptors as well as effectors of cell proliferation and migration in Nck1 and Nck2 knockout mice, in line with the distinct phenotypes observed at 5 and 8 weeks. Together these studies reveal a requirement for NCK proteins in mammary gland morphogenesis, and suggest that deregulation of Nck expression could drive breast cancer progression and metastasis.

Sex Differences in Glomerular Protein Expression and Effects of Soy-Based Diet on Podocyte Signaling.

Background: Kidney disease is a major public health issue arising from loss of glomerular podocyte function, and there are considerable sex differences in its prognosis. Evidence suggests a renoprotective effect of estrogen and soy diet-derived phytoestrogens, although the molecular basis for this is poorly understood. Objective: Here, we aim to assess sex differences in expression of key proteins associated with podocyte survival and determine the effects of dietary soy on glomerular and podocyte signaling. Methods: Male and female FVB mice were fed control, low (1%), and high (20%) doses of isolated soy protein (ISP) in utero and until 100 days of age. Spot urine was collected to measure proteinuria and isolated glomeruli were used to quantify activated and total levels of nephrin, Akt, and ERK1/2. To investigate protective effects of specific soy phytoestrogens, cultured podocytes were treated with or without daidzein and subject to control or high glucose as a model of podocyte injury. Results: Nephrin and Akt were elevated at baseline in glomeruli from females compared to males. Both sexes that were fed 1% and 20% ISP displayed robust increases in total glomerular Akt compared to controls, and these effects were more prominent in females. A similar trend at both doses in both sexes was observed with activated Akt and total nephrin. Notably, males exclusively showed increased phosphorylation of nephrin and extracellular signal-regulated kinase (ERK) at the 1% ISP dose; however, no overt changes in urinary albumin excretion or podocin levels were observed, suggesting that the soy diets did not impair podocyte function. Finally, in cultured male and female podocytes, daidzein treatment suppressed high glucose-induced ERK activation. Conclusions: Together, our findings reveal a putative mechanism to explain the protective influence of sex on kidney disease progression, and they provide further evidence to support a beneficial role for dietary soy in preserving glomerular function.

Contexte: L'insuffisance rénale est un problème majeur de santé publique résultant d'une perte de fonction des podocytes glomérulaires, et son pronostic diffère selon le sexe. Bien que le fondement moléculaire en soit mal compris, des données suggèrent que les œstrogènes et des phytoestrogènes dérivés du soja alimentaire auraient un effet néphroprotecteur. Objectifs: Évaluer les différences selon le sexe dans l'expression des protéines clés associées à la survie des podocytes, et déterminer les effets du soja alimentaire sur la signalisation glomérulaire et les podocytaire. Méthodologie: Des souris FVB mâles et femelles ont reçu un régime alimentaire témoin ou un regime à faible dose (1 %) ou à dose élevée (20 %) de protéines de soja isolées (PSI) in utero et jusqu'à l'âge de 100 jours. Des échantillons aléatoires d'urine ont été recueillis pour mesurer la protéinurie et des glomérules isolés ont été utilisés pour quantifier les niveaux activés et totaux de néphrine, d'Akt et d'ERK1/2. Pour évaluer l'effet protecteur de certains phytoestrogènes du soja, des podocytes cultivés ont été traités avec ou sans daidzéine et soumis à une dose témoin ou à une dose élevée de glucose comme modèle de lésion podocytaire. Résultats: Les taux initiaux de néphrine et d'Akt étaient plus élevés dans les glomérules des souris femelles. Les souris mâles et femelles nourries avec des doses de 1 % et de 20 % de PSI ont montré des augmentations significatives de l'Akt glomérulaire totale par rapport aux témoins, et ces effets étaient plus importants chez les femelles. Une tendance semblable a été observée chez les deux sexes et pour les deux doses en ce qui concerne l'Akt activée et la néphrine totale. Seuls les mâles ont montré une augmentation de la phosphorylation de la néphrine et de l'ERK à 1 % de PSI; aucun changement manifeste n'a cependant été observé dans l'excrétion urinaire d'albumine ou dans le taux de podocine, ce qui suggère que le soja alimentaire n'a pas altéré la fonction des podocytes. Dans les podocytes cultivés, tant mâles que femelles, le traitement à la daidzéine a inhibé l'activation de l'ERK induite par une forte dose de glucose. Conclusion: Ensemble, nos résultats révèlent un mécanisme putatif pouvant expliquer l'effet protecteur du sexe du patient sur la progression de l'insuffisance rénale. Ces résultats fournissent des preuves supplémentaires soutenant l'hypothèse d'un rôle bénéfique du soja alimentaire dans la préservation de la fonction glomérulaire.

Elevated Expression of miR-200c/141 in MDA-MB-231 Cells Suppresses MXRA8 Levels and Impairs Breast Cancer Growth and Metastasis In Vivo.

Breast cancer cells with mesenchymal characteristics, particularly the claudin-low subtype, express extremely low levels of miR-200s. Therefore, this study examined the functional impact of restoring miR-200 expression in a human claudin-low breast cancer cell line MDA-MB-231. MDA-MB-231 cells were stably transfected with a control vector (MDA-231EV) or the miR-200c/141 cluster (MDA-231c141). Injection of MDA-231c141 cells into the 4th mammary gland of NCG mice produced tumors that developed significantly slower than tumors produced by MDA-231EV cells. Spontaneous metastasis to the lungs was also significantly reduced in MDA-231c141 cells compared to MDA-231EV cells. RNA sequencing of MDA-231EV and MDA-231c141 tumors identified genes including MXRA8 as being downregulated in the MDA-231c141 tumors. MXRA8 was further investigated as elevated levels of MXRA8 were associated with reduced distant metastasis free survival in breast cancer patients. Quantitative RT-PCR and Western blotting confirmed that MXRA8 expression was significantly higher in mammary tumors induced by MDA-231EV cells compared to those induced by MDA-231c141 cells. In addition, MXRA8 protein was present at high levels in metastatic tumor cells found in the lungs. This is the first study to implicate MXRA8 in human breast cancer, and our data suggests that miR-200s inhibit growth and metastasis of claudin-low mammary tumor cells in vivo through downregulating MXRA8 expression.

Transgenic overexpression of the miR-200b/200a/429 cluster inhibits mammary tumor initiation.

The miR-200 family consists of five members expressed as two clusters: miR-200c/141 cluster and miR-200b/200a/429 cluster. In the mammary gland, miR-200s maintain epithelial identity by decreasing the expression of mesenchymal markers leading to high expression of epithelial markers. While the loss of miR-200s is associated with breast cancer growth and metastasis the impact of miR-200 expression on mammary tumor initiation has not been investigated. Using mammary specific expression of the miR-200b/200a/429 cluster in transgenic mice, we found that elevated expression miR-200s could almost completely prevent mammary tumor development. Only 1 of 16 MTB-IGFIRba429 transgenic mice (expressing both the IGF-IR and miR-200b/200a/429 transgenes) developed a mammary tumor while 100% of MTB-IGFIR transgenic mice (expressing only the IGF-IR transgene) developed mammary tumors. RNA sequencing, qRT-PCR, and immunohistochemistry of mammary tissue from 55-day old mice found Spp1, Saa1, and Saa2 to be elevated in mammary tumors and inhibited by miR-200b/200a/429 overexpression. This study suggests that miR-200s could be used as a preventative strategy to protect women from developing breast cancer. One concern with this approach is the potential negative impact miR-200 overexpression may have on mammary function. However, transgenic overexpression of miR-200s, on their own, did not significantly impact mammary ductal development indicating the miR-200 overexpression should not significantly impact mammary function. Thus, this study provides the initial foundation for using miR-200s for breast cancer prevention and additional studies should be performed to identify strategies for increasing mammary miR-200 expression and determine whether miR-200s can prevent mammary tumor initiation by other genetic alterations.

The miR-200 family in normal mammary gland development.

The miR-200 family of microRNAs plays a significant role in inhibiting mammary tumor growth and progression, and its members are being investigated as therapeutic targets. Additionally, if future studies can prove that miR-200s prevent mammary tumor initiation, the microRNA family could also offer a preventative strategy. Before utilizing miR-200s in a therapeutic setting, understanding how they regulate normal mammary development is necessary. No studies investigating the role of miR-200s in embryonic ductal development could be found, and only two studies examined the impact of miR-200s on pubertal ductal morphogenesis. These studies showed that miR-200s are expressed at low levels in virgin mammary glands, and elevated expression of miR-200s have the potential to impair ductal morphogenesis. In contrast to virgin mammary glands, miR-200s are expressed at high levels in mammary glands during late pregnancy and lactation. miR-200s are also found in the milk of several mammalian species, including humans. However, the relevance of miR-200s in milk remains unclear. The increase in miR-200 expression in late pregnancy and lactation suggests a role for miR-200s in the development of alveoli and/or regulating milk production. Therefore, studies investigating the consequence of miR-200 overexpression or knockdown are needed to identify the function of miR-200s in alveolar development and lactation.

Isolated Soy Protein Promotes Mammary Tumor Development Induced by the Type I Insulin-like Growth Factor Receptor in Transgenic Mice.

Studies suggest consuming soy may protect women from breast cancer. In this study, lifetime exposure to 20%, 5% and 1% ISP in MTB-IGFIR mice (mammary-specific expression of IGF-IR) were evaluated to determine whether ISP could protect against mammary tumorigenesis. MTB-IGFIR mice fed ISP diets displayed increased mammary tumor incidence and reduced tumor latency compared to mice fed 20% casein. To evaluate whether a diet containing a less refined form of soy could protect against mammary tumor development MTB-IGFIR mice were fed Teklad 2018 (contains soybean meal). MTB-IGFIR mice fed the Teklad 2018 diet were completely protected against mammary tumor development. To determine whether dietary ISP was sufficient to induce mammary tumorigenesis, MTB-IGFIR mice were fed Teklad 2018ISP (soybean meal of Teklad 2018 was replaced with an equivalent amount of ISP). Only two of 10 MTB-IGFIR mice fed Teklad 2018ISP developed mammary tumors. This study demonstrates the complex interaction between soy and other dietary components in modifying mammary tumor development.

Nidogen 1 regulates proliferation and migration/invasion in murine claudin-low mammary tumor cells.

Nidogen 1 (NID1) is a glycoprotein found in basement membranes involved in cross-linking collagen IV and laminin. The role of NID in breast cancer has only been evaluated in a small number of studies and the findings of these studies have been inconsistent. Our previous work revealed that highly tumorigenic murine mammary tumor cells express high levels of Nid1 while weakly tumorigenic mammary tumor cells express low levels of Nid1. To investigate Nid1, two stable knockdown lines were created, and Nid1 knockdown was confirmed at both the mRNA and protein level. Nid1 knockdown significantly reduced cell proliferation and migration/invasion and these reductions in proliferation and migration/invasion could be rescued by conditioned media containing NID1 protein. The reduced migration/invasion observed in the Nid1 knockdown cells was not associated with significant alterations in the epithelial gene Cdh1 or the mesenchymal genes Snai1, Snai2, Twist1, Twist2, Zeb1 and Zeb2. Therefore, suppression of Nid1 expression reduces proliferation and migration/invasion in claudin-low murine mammary tumor cells.

Polycomb repressor complex 2 function in breast cancer (Review).

Epigenetic modifications are important contributors to the regulation of genes within the chromatin. The polycomb repressive complex 2 (PRC2) is a multi­subunit protein complex that is involved in silencing gene expression through the trimethylation of lysine 27 at histone 3 (H3K27me3). The dysregulation of this modification has been associated with tumorigenicity through the increased repression of tumour suppressor genes via condensing DNA to reduce access to the transcription start site (TSS) within tumor suppressor gene promoters. In the present review, the core proteins of PRC2, as well as key accessory proteins, will be described. In addition, mechanisms controlling the recruitment of the PRC2 complex to H3K27 will be outlined. Finally, literature identifying the role of PRC2 in breast cancer proliferation, apoptosis and migration, including the potential roles of long non­coding RNAs and the miR­200 family will be summarized as will the potential use of the PRC2 complex as a therapeutic target.

Rodent Models Assessing Mammary Tumor Prevention by Soy or Soy Isoflavones.

While epidemiological studies performed in Asian countries generally show that high levels of dietary soy are associated with reduced breast cancer risk, studies in Western countries have typically failed to show this correlation. In an attempt to model the preventative actions of soy on mammary tumor development, rodent models have been employed. Thirty-four studies were identified that evaluated the impact of soy products or purified soy isoflavones on mammary tumor initiation (studies evaluating established mammary tumors or mammary tumor cell lines were not included) and these studies were separated into mammary tumors induced by chemical carcinogens or transgenic expression of oncogenes based on the timing of soy administration. Regardless of when soy-based diets or purified isoflavones were administered, no consistent protective effects were observed in either carcinogen-induced or oncogene-induced mammary tumors. While some studies demonstrated that soy or purified isoflavones could reduce mammary tumor incidence, other studies showed either no effect or tumor promoting effects of soy products or isoflavones. Most importantly, only five studies found a decrease in mammary tumor incidence and six studies observed a decrease in tumor multiplicity, two relevant measures of the tumor preventative effects of soy or isoflavones. The variable outcomes of the studies examined were not completely surprising given that few studies employed the same experimental design. Future studies should be carefully designed to more accurately emulate soy consumption observed in Asian cultures including lifetime exposure to less refined soy products and potentially the incorporation of multigenerational feeding studies.

Integrative analysis of copy number and gene expression data identifies potential oncogenic drivers that promote mammary tumor recurrence.

Tumor recurrence represents a significant clinical challenge in the treatment and management of breast cancer. To investigate whether copy number aberrations (CNAs) facilitate the re-emergence of tumor growth from residual disease, we performed array comparative genomic hybridization on primary and recurrent mammary tumors from an inducible mouse model of type-I insulin-like growth factor receptor driven breast cancer. This genome-wide analysis revealed primary and recurrent tumors harbored distinct CNAs with relapsed tumors containing an increased number of gene-level gains and losses. Remarkably, high-level CNAs detected in primary tumors were largely devoid of annotated cancer genes while the vast majority of recurrent tumors harbored at least one CNA containing a known oncogene or tumor suppressor. Specifically, 38% of recurrent tumors carried gains at 6qA2 and 9qA2 which encode the Met and Yap1 oncogenes, respectively. The most frequent CNA, occurring in 63% of recurrent tumors, was a focal deletion at 4qC5 involving the Cdkn2a/b tumor suppressor genes. Integrative analysis revealed positive correlations between gene copy number and mRNA expression suggesting Met, Yap1, and Cdkn2a/b may serve as potential drivers that promote tumor recurrence. Accordingly, cross-species analysis revealed gene-level murine CNAs were present in a subset of human breast cancers with high MET and YAP1 mRNA predictive of decreased relapse-free survival in basal-like breast cancers. Together, these findings indicate that tumor recurrence is facilitated by the acquisition of CNAs with oncogenic potential and provide a framework to dissect the molecular mechanisms that mediate tumor escape from dormancy.

Comparative mRNA and miRNA transcriptome analysis of a mouse model of IGFIR-driven lung cancer.

Mouse models of cancer play an important role in elucidating the molecular mechanisms that contribute to tumorigenesis. The extent to which these models resemble one another and their human counterparts at the molecular level is critical in understanding tumorigenesis. In this study, we carried out a comparative gene expression analysis to generate a detailed molecular portrait of a transgenic mouse model of IGFIR-driven lung cancer. IGFIR-driven tumors displayed a strong resemblance with established mouse models of lung adenocarcinoma, particularly EGFR-driven models highlighted by elevated levels of the EGFR ligands Ereg and Areg. Cross-species analysis revealed a shared increase in human lung adenocarcinoma markers including Nkx2.1 and Napsa as well as alterations in a subset of genes with oncogenic and tumor suppressive properties such as Aurka, Ret, Klf4 and Lats2. Integrated miRNA and mRNA analysis in IGFIR-driven tumors identified interaction pairs with roles in ErbB signaling while cross-species analysis revealed coordinated expression of a subset of conserved miRNAs and their targets including miR-21-5p (Reck, Timp3 and Tgfbr3). Overall, these findings support the use of SPC-IGFIR mice as a model of human lung adenocarcinoma and provide a comprehensive knowledge base to dissect the molecular pathogenesis of tumor initiation and progression.

The miR-200b/200a/429 cluster prevents metastasis and induces dormancy in a murine claudin-low mammary tumor cell line.

The miR-200 family of microRNAs consisting of miR-141, miR-200a, miR-200b, miR-200c and miR-429 are emerging as important regulators of breast cancer progression. This family of microRNAs maintain mammary epithelial identity and downregulation of miR-200 expression has been associated with epithelial-to-mesenchymal transition in mammary tumors. Therefore, re-expression of one or more miR-200 family members in mammary tumor cells with mesenchymal characteristics may restore an epithelial phenotype including growth and metastasis suppression. To test this hypothesis, the miR-200b/200a/429 cluster was re-expressed in a murine claudin-low cell line, RJ423. Re-expression of the miR-200b/200a/429 cluster in RJ423 cells significantly suppressed the expression of Vim, Snai1, Twist1, Twist2 and Zeb1, reverted RJ423 cells to a more epithelial morphology and significantly inhibited proliferation in vitro. Moreover, the miR-200b/200a/429 cluster prevented lung metastasis in an experimental metastasis model and although tumor initiation was not prevented, re-expression of the miR-200b/200a/429 cluster induced a dormancy-like state where mammary tumors failed to grow beyond ~150 mm3 or grew extremely slowly following intra-mammary injection. These dormant tumors contained elevated levels of collagen and were highly vascularized. Therefore, re-expression of the miR-200b/200a/429 cluster in the claudin-low mammary tumor cell line, RJ423, is sufficient to alter cell morphology, impair metastasis and induce tumor dormancy.

A-674563, a putative AKT1 inhibitor that also suppresses CDK2 activity, inhibits human NSCLC cell growth more effectively than the pan-AKT inhibitor, MK-2206.

AKT is a serine-threonine kinase implicated in tumorigenesis as a central regulator of cellular growth, proliferation, survival, and metabolism. Activated AKT is commonly overexpressed in non-small cell lung cancer (NSCLC) and accordingly AKT inhibitors are under clinical investigation for NSCLC treatment. Thus far, the AKT inhibitors being evaluated broadly target all three (1-3) AKT isoforms but recent evidence suggests opposing roles in lung tumorigenesis where loss of Akt1 inhibits while the loss of Akt2 enhances lung tumor development. Based on these findings, we hypothesized that selective inhibition of AKT-1 would be a more effective therapeutic strategy than pan-AKT inhibition for NSCLC treatment. Using six NSCLC cell lines, we found that the AKT-1 inhibitor, A-674563, was significantly more effective at reducing NSCLC cell survival relative to the pan-AKT inhibitor MK-2206. Comparison of the downstream effects of the inhibitors suggests that altered cell cycle progression and off-target CDK2 inhibition are likely vital to the improved efficacy of A-674563 over MK-2206.

BMS-754807 is cytotoxic to non-small cell lung cancer cells and enhances the effects of platinum chemotherapeutics in the human lung cancer cell line A549.

BACKGROUND: Despite advances in targeted therapy for lung cancer, survival for patients remains poor and lung cancer remains the leading cause of cancer-related deaths worldwide. The type I insulin-like growth factor receptor (IGF-IR) has emerged as a potential target for lung cancer treatment, however, clinical trials to date have provided disappointing results. Further research is needed to identify if certain patients would benefit from IGF-IR targeted therapies and the ideal approach to incorporate IGF-IR targeted agents with current therapies. METHODS: The dual IGF-IR/insulin receptor inhibitor, BMS-754807, was evaluated alone and in combination with platinum-based chemotherapeutics in two human non-small cell lung cancer (NSCLC) cell lines. Cell survival was determined using WST-1 assays and drug interaction was evaluated using Calcusyn software. Proliferation and apoptosis were determined using immunofluorescence for phospho-histone H3 and cleaved caspase 3, respectively. RESULTS: Treatment with BMS-754807 alone reduced cell survival and wound closure while enhancing apoptosis in both human lung cancer cell lines. These effects appear to be mediated through IGF-IR/IR signaling and, at least in part, through the PI3K/AKT pathway as administration of BMS-754807 to A549 or NCI-H358 cells significantly suppressed IGF-IR/IR and AKT phosphorylation. In addition of BMS-754807 enhanced the cytotoxic effects of carboplatin or cisplatin in a synergistic manner when given simultaneously to A549 cells. CONCLUSIONS: BMS-754807 may be an effective therapeutic agent for the treatment of NSCLC, particularly in lung cancer cells expressing high levels of IGF-IR.

Unique roles of Akt1 and Akt2 in IGF-IR mediated lung tumorigenesis.

AKT is a serine-threonine kinase that becomes hyperactivated in a number of cancers including lung cancer. Based on AKT's association with malignancy, molecules targeting AKT have entered clinical trials for solid tumors including lung cancer. However, the AKT inhibitors being evaluated in clinical trials indiscriminately inhibit all three AKT isoforms (AKT1-3) and it remains unclear whether AKT isoforms have overlapping or divergent functions. Using a transgenic mouse model where IGF-IR overexpression drives lung tumorigenesis, we found that loss of Akt1 inhibited while loss of Akt2 enhanced lung tumor development. Lung tumors that developed in the absence of Akt2 were less likely to appear as discrete nodules and more frequently displayed a dispersed growth pattern. RNA sequencing revealed a number of genes differentially expressed in lung tumors lacking Akt2 and five of these genes, Actc1, Bpifa1, Mmp2, Ntrk2, and Scgb3a2 have been implicated in human lung cancer. Using 2 human lung cancer cell lines, we observed that a selective AKT1 inhibitor, A-674563, was a more potent regulator of cell survival than the pan-AKT inhibitor, MK-2206. This study suggests that compounds selectively targeting AKT1 may prove more effective than compounds that inhibit all three AKT isoforms at least in the treatment of lung adenocarcinoma.

High levels of dietary soy decrease mammary tumor latency and increase incidence in MTB-IGFIR transgenic mice.

BACKGROUND: Epidemiologic data indicates that Asian diets, which are high in soy protein, reduce a women's risk of developing breast cancer. However, it has been difficult to dissociate the benefits of soy from other variables including environmental and lifestyle factors. Since prospective studies in humans would take decades to complete, rodent models provide a valuable research alternative. METHODS: In this study, MTB-IGFIR transgenic mice, which develop mammary tumors resulting from overexpression of the type I insulin-like growth factor receptor (IGF-IR), were utilized. MTB-IGFIR mice were fed a soy-based or casein-based diet throughout all stages of development to reflect soy exposure in Asian cultures. Mammary tumors were initiated at 2 different developmental stages by commencing IGF-IR transgene expression either during puberty or in adult mice. RESULTS: MTB-IGFIR mice fed a soy-based diet displayed increased tumor incidence and accelerated tumor onset compared to MTB-IGFIR mice fed a casein diet. Two markers of estrogen receptor signaling, Pgr and Areg, were elevated in mammary tissue from mice fed the soy diet compared to mice fed the casein diet suggesting that high levels of soy may promote mammary tumor development through acting as an estrogen receptor agonist. Mammary tumors from mice fed a soy diet more frequently expressed metaplastic markers such as cytokeratins 5 and 14 as well as p63 and displayed reduced lung metastases compared to mammary tumors from mice fed a casein diet. CONCLUSIONS: Diets consisting of very high levels of soy protein promote mammary tumor development and decrease tumor latency possibly through activating estrogen receptor signaling. Additional studies are required to determine whether a more moderate amount of dietary soy can inhibit oncogene-induced mammary tumorigenesis.

IGF-IR mediated mammary tumorigenesis is enhanced during pubertal development.

Although breast cancer typically develops in women over the age of 40, it remains unclear when breast cancer initiating events occur or whether the mammary gland is particularly susceptible to oncogenic transformation at a particular developmental stage. Using MTB-IGFIR transgenic mice that overexpress type I insulin-like growth factor receptor (IGF-IR) in a doxycycline-inducible manner, mammary tumorigenesis was initiated at different developmental stages. Tumor multiplicity was significantly increased while tumor latency was significantly decreased when the IGF-IR transgene was expressed during pubertal development compared to post-pubertal transgene expression. Moreover, metastatic spread of mammary tumors to the lungs was approximately twice as likely when IGF-IR was overexpressed in pubertal mice compared to post-pubertal mice. In addition, engraftment of pubertal MTB-IGFIR mammary tissue into cleared mammary fat pads of pubertal hosts produced tumors more frequently and faster than engraftment into adult hosts. These experiments show that the mammary microenvironment created during puberty renders mammary epithelial cells particularly susceptible to transformation.

Inhibition of proliferation and migration of luminal and claudin-low breast cancer cells by PDGFR inhibitors.

BACKGROUND: Platelet-derived growth factors (PDGFs) bind to two receptors, PDGFRα and PDGFRß to mediate cell proliferation, migration and survival. Although epithelial cells typically do not express high levels of PDGFRs, their expression has been reported to increase in breast cancer cells that have undergone epithelial to mesenchymal transition. METHODS: PDGFR signaling was inhibited using Sunitinib malate, Imatinib mesylate or Regorafenib in murine and human luminal-like and claudin-low mammary tumor cell lines or Masitinib in only the human cell lines. A scratch wound assay was used to assess tumor cell migration while immunofluorescence for phosphorylated histone H3 or cleaved caspase 3 was used to determine tumor cell proliferation and apoptosis, respectively. RESULTS: Sunitinib and Regorafenib, but not Imatinib, were capable of significantly inhibiting the migration of both murine and human luminal-like and claudin-low breast cancer cells while Masitinib inhibited migration in both human breast cancer cell lines. Sunitinib but not Regorafenib or Imatinib also significantly suppressed tumor cell proliferation in all four cell lines tested while Masitinib had no significant effect on human breast cancer cell proliferation. None of the PDGFR inhibitors consistently regulated mammary tumor cell apoptosis. CONCLUSION: Sunitinib, Regorafenib and Masitinib may prove clinically useful in inhibiting breast cancer cell migration and metastasis while only Sunitinib (and possibly Regorafenib in some breast cancer subtypes) is effective at inhibiting both migration and proliferation of breast cancer cells.