Polyoma Middle T Oncoprotein Research Articles

Abstract 5114: The role of amphiregulin in mammary gland development and breast cancer

Abstract Breast cancer is the most prevalent cancer in women worldwide and afflicts more than 3.1 million women in the US alone. The epidermal growth factor receptor (EGFR) is frequently overexpressed in breast cancer and is associated with poor clinical outcome. Several therapies involving anti-EGFR agents have been developed to target breast cancer. However, clinical trials have shown mixed outcomes with these therapies. Amphiregulin (AREG), a ligand of the epidermal growth factor receptor (EGFR), is enriched in invasive breast cancer, particularly ERα-positive breast tumors. In normal physiology, AREG mediates estrogen-induced ductal morphogenesis during mammary gland development. In the absence of AREG, the ductal tree fails to elongate and remains rudimentary throughout adulthood. The structure of a healthy mammary duct consists of luminal cells surrounded by a layer of myoepithelial cells. Studies of mammary gland transplantations suggest that a marker of myoepithelial cells (keratin 14, K14) is only expressed if AREG is present in the mammary epithelium. Multiple studies have provided evidence that myoepithelial cells are natural tumor suppressors. During tumor growth, the disappearance of myoepithelial cells indicates progression to invasive carcinoma. Therefore we hypothesize that AREG plays an important role in maintenance of the maintenance of myoepithelial cells in the mammary epithelium and loss of AREG promotes tumor progression as a result of an early loss of myoepithelial cells. In the mammary glands of AREG WT and AREG KO mice without the PyMT transgene, we have found that mammary ducts in AREG KO mice have significantly fewer myoepithelial cells. Immunofluorescent staining of K14 has revealed a thinner and discontinuous myoepithelial layer in the AREG KO mice. To investigate the role of AREG in breast cancer development and progression, we have utilized a spontaneous mammary tumor mouse model where oncogenesis is driven by expression of the polyoma middle T oncoprotein (PyMT) through the mouse mammary tumor virus (MMTV) promoter. We have crossed AREG knockout mice with the MMTV-PyMT mice to generate AREG KO mice that spontaneously form mammary tumors (AREG KO PyMT). The absence of AREG in both the mammary epithelial and stromal compartments accelerates tumor growth. By 12 weeks of age, tumors of AREG KO PyMT mice are significantly larger than that of AREG WT PyMT mice. AREG KO tumors are histologically less necrotic, apoptotic, and progressed than AREG WT tumors. Furthermore, AREG KO tumors show higher staining of PyMT by immunofluorescence. Together, these results suggest that loss of AREG stimulates tumor initiation but inhibits progression due to fewer myoepithelial cells beginning during development. Citation Format: Serena Mao, Minji Park, Ramon Cabrera, John Christin, George Karagiannis, Wenjun Guo, John Condeelis, Jeffrey Segall. The role of amphiregulin in mammary gland development and breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5114.

Abstract 1844: Investigating the role of amphiregulin in breast cancer

Abstract Breast cancer is the most prevalent cancer in women worldwide and afflicts more than 3.1 million women in the US alone. The epidermal growth factor receptor (EGFR) is frequently overexpressed in breast cancer and is associated with poor clinical outcome. Several therapies involving anti-EGFR agents have been developed to target breast cancer. However, clinical trials have shown mixed outcomes with these therapies. Amphiregulin (AREG) is a ligand for EGFR that mediates estrogen-induced mammary ductal morphogenesis during development. Expression of AREG is enriched in invasive breast carcinomas, particularly ERα-positive breast tumors. Studies have shown that AREG increases the in vitro invasiveness of breast cancer cells. Furthermore, suppression of AREG expression in transformed human breast epithelial cells reduces tumor formation when injected in nude mice. These observations suggest that AREG plays an important role in breast cancer development and progression. To investigate the role of AREG in breast cancer development and progression, we utilized a spontaneous mammary tumor mouse model where oncogenesis is driven by expression of the polyoma middle T oncoprotein (PyMT) through the mouse mammary tumor virus (MMTV) promoter. We have crossed AREG knockout mice with the MMTV-PyMT mice to generate AREG KO mice that spontaneously form mammary tumors. Tumors in AREG KO PyMT mice initially grow more quickly but eventually reach 2 cm in diameter at a relatively time as compared to tumors formed in AREG WT PyMT mice. Histologically, AREG KO tumors appear more differentiated and display a greater proportion of tumor stroma. This prompted us to further hypothesize that AREG may have diverse effects in the tumoral or stromal compartment of developing tumors. To individually assess the effects of AREG knockout in the tumor cells and the stroma, we utilized a transplantation model in which we prepared tumor chunks from AREG WT and KO PyMT tumors and transplanted them into AREG WT and KO mice. Through this model, we have found that AREG expression in the tumor has a greater impact on tumor growth and progression than the tumor stroma. Regardless of the AREG status of the recipient mice, AREG KO tumors display a significant growth impairment and reduced transplant efficiency. In summary, these results demonstrate that AREG is important for mammary tumor progression. Citation Format: Serena P. Chiang, George S. Karagiannis, John S. Condeelis, Jeffrey E. Segall. Investigating the role of amphiregulin in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1844. doi:10.1158/1538-7445.AM2017-1844

Cystatin C deficiency suppresses tumor growth in a breast cancer model through decreased proliferation of tumor cells.

Cysteine cathepsins are proteases that, in addition to their important physiological functions, have been associated with multiple pathologies, including cancer. Cystatin C (CstC) is a major endogenous inhibitor that regulates the extracellular activity of cysteine cathepsins. We investigated the role of cystatin C in mammary cancer using CstC knockout mice and a mouse model of breast cancer induced by expression of the polyoma middle T oncoprotein (PyMT) in the mammary epithelium. We showed that the ablation of CstC reduced the rate of mammary tumor growth. Notably, a decrease in the proliferation of CstC knockout PyMT tumor cells was demonstrated ex vivo and in vitro, indicating a role for this protease inhibitor in signaling pathways that control cell proliferation. An increase in phosphorylated p-38 was observed in CstC knockout tumors, suggesting a novel function for cystatin C in cancer development, independent of the TGF-β pathway. Moreover, proteomic analysis of the CstC wild-type and knockout PyMT primary cell secretomes revealed a decrease in the levels of 14-3-3 proteins in the secretome of knock-out cells, suggesting a novel link between cysteine cathepsins, cystatin C and 14-3-3 proteins in tumorigenesis, calling for further investigations.

Multimodal In Vivo Imaging of Tumorigenesis and Response to Chemotherapy in a Transgenic Mouse Model of Mammary Cancer.

PurposeTransgenic mice expressing the polyoma middle T oncoprotein (PyMT) in the mammary epithelium were explored by multimodal imaging to monitor longitudinally spontaneous tumor growth and response to chemotherapy.ProceduresPositron emission tomography (PET) with 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) and 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT), single photon emission tomography (SPECT) with [99mTc]TcO4 ([99mTc]TEC), X-ray computed tomography, and fluorescent confocal endomicroscopy (FCE) images were acquired during tumor progression in female PyMT mice. Imaging with [18F]FDG and [99mTc]TEC was also performed in untreated, doxorubicin-treated, and docetaxel-treated PyMT mice. Total tumor volumes were quantified. Tumors were collected and macroscopic and histological examinations were performed.ResultsAll PyMT mice developed multifocal tumors of the mammary epithelium that became palpable at 8 weeks of age (W8). Computed tomography (CT) detected tumors at W14, while a clear tumoral uptake of [99mTc]TEC and [18F]FDG was present as early as W6 and W8, respectively. No contrast between mammary tumors and surrounding tissue was observed at any stage with [18F]FLT. FCE detected an angiogenic switch at W10. Lung metastases were not clearly evidenced by imaging. Doxorubicin and docetaxel treatments delayed tumor growth, as shown by [18F]FDG and [99mTc]TEC, but tumor growth resumed upon treatment discontinuation. Tumor growth fitted an exponential model with time constant rates of 0.315, 0.145, and 0.212 week−1 in untreated, doxorubicin, and docetaxel groups, respectively.ConclusionsMolecular imaging of mammary tumors in PyMT is precocious, precise, and predictive. [18F]FDG-PET and [99mTc]TEC SPECT monitor tumor response to chemotherapy.Electronic supplementary materialThe online version of this article (doi:10.1007/s11307-015-0916-7) contains supplementary material, which is available to authorized users.

Abstract 4019: Examining mechanisms by which macrophages enhance intravasation in breast cancer

Abstract Breast cancer is the most common invasive malignancy and a leading cause of cancer-associated deaths in women worldwide. Metastasis is the major cause of morbidity and mortality in women afflicted with breast cancer. In the metastatic process, intravasation is an important step and largely dependent on interactions between tumor cells and stromal cells in the tumor microenvironment (TME). Tumor-associated macrophages (TAMs), an integral component of the TME, have been found to associate and co-migrate with tumor cells during intravasation. Invasive TAMs that co-migrate with tumor cells in response to epidermal growth factor display a unique transcriptome in comparison to the general TAM population found in the TME. Furthermore, intercellular signaling between tumor cells and TAMs generates a positive feedback loop that promotes tumor cell invasion and intravasation. Chemokines play a major role in cell migration and are an important component of the TME. In particular, chemokine (C-X-C motif) receptor 2 (CXCR2) has been shown to be important in tumor growth and angiogenesis. MIF and IL8, two ligands of CXCR2, correlate with poor outcome in breast cancer. Our objective is to elucidate the role of macrophage CXCR2 in tumor cell intravasation. We have used flow cytometry to characterize the basal expression of CXCR2 in a murine macrophage cell line, BAC-1.2F5 (BAC). We have also analyzed mRNA expression of CXCR2 in BAC and primary TAMs isolated from a murine model of breast cancer, the polyoma middle T oncoprotein (PyMT), by qRT-PCR. To test the functional significance of CXCR2 in intravasation, we have examined the effects of an inhibitor of CXCR2 on the ability of tumor cells to migrate through the endothelium using an in vitro transendothelial migration assay (iTEM). In this assay, MDA-MB-231 tumor cells, a human breast cancer cell line, are co-cultured with BAC cells and subsequently placed in an environment that is in contact with the basal surface of endothelial cells. We have observed that BAC cells produce CXCR2 mRNA and express surface CXCR2. We have also found elevated expression of CXCR2 in invasive TAMs as compared to the general TAM population. When MDA-MB-231 cells are co-cultured with BAC cells in the iTEM assay in the presence of SB265610, an inhibitor of CXCR2, we have found a significant reduction (p<10-4) in iTEM. Together, these results suggest that cellular signaling through macrophage CXCR2 is critical to tumor cell intravasation. Citation Format: Serena Chiang, Zhenni Zhou, Jeffrey E. Segall. Examining mechanisms by which macrophages enhance intravasation in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4019. doi:10.1158/1538-7445.AM2015-4019

Exercise training in transgenic mice is associated with attenuation of early breast cancer growth in a dose-dependent manner.

Epidemiological research suggests that regular physical activity confers beneficial effects that mediate an anti-tumor response and may reduce cancer recurrence. It is unclear what amount of physical activity is necessary to exert such a protective effect and what mechanisms are involved. We investigated the effects of voluntary wheel running on tumor progression and cytokine gene expression in the transgenic polyoma middle T oncoprotein (PyMT) mouse model of invasive breast cancer. Runners showed significantly reduced tumor sizes compared with non-runners after 3 weeks of running (p≤0.01), and the greater the running distance the smaller the tumor size (Pearson's r = −0.61, p≤0.04, R2 = 0.38). Mice running greater than 150 km per week had a significantly attenuated tumor size compared with non-runners (p≤0.05). Adipose tissue mass was inversely correlated with tumor size in runners (Pearson's r = −0.77, p = 0.014) but not non-runners. Gene expression of CCL22, a cytokine associated with recruitment of immunosuppressive T regulatory cells, was decreased in tumors of runners compared to non-runners (p≤0.005). No differences in tumor burden or metastatic burden were observed between runners and non-runners after ten weeks of running when the study was completed. We conclude that voluntary wheel running in PyMT mice correlates with an attenuation in tumor progression early during the course of invasive breast cancer. This effect is absent in the later stages of overwhelming tumor burden even though cytokine signaling for immunosuppressive regulatory T cells was down regulated. These observations suggest that the initiation of moderate exercise training for adjunctive therapeutic benefit early in the course of invasive breast cancer should be considered for further investigation.

Abstract PL03-01: Macrophage diversity promotes tumor progression and metastasis

Abstract The tumor microenvironment represents a complex and changing ecology of many different cell types whose interactions determines the overall malignancy of the tumor. This environment consists of tumor cells harboring oncogenic and tumor suppressor mutations together with normal cells that include resident ones and those recruited from the bone marrow. In this latter class myeloid cells and especially macrophages, the subject of our studies, are particularly well represented. Associative clinical studies have indicated that the abundance of macrophages or overexpression of their signaling pathways is often an indicator of poor prognosis in a wide variety of cancers. These data suggests a pro-tumoral role for tumor-associated macrophages. This hypothesis that macrophages promoted malignancy is supported by a significant number of studies using genetic or pharmacological ablation of macrophages or their signaling pathways. In our studies, we used genetic ablation of macrophages in the model of breast cancer caused by the mammary epithelial expression of the Polyoma Middle T oncoprotein (PyMT). This ablation resulted in a slowing of tumor progression to malignancy and an inhibition of metastasis. We and our collaborators have identified several traits that macrophages confer in the primary tumor that account for their enhancement of malignancy. These are in the promotion of angiogenesis, stimulation of tumor cell invasion, migration and intravasation. At the metastatic site we have also shown that macrophages promote tumor cell extravasation and subsequent growth. Macrophage isolation and characterization from PyMT tumors and their metastatic sites suggest that each of these activities is regulated by a distinct subsets of macrophages that nevertheless express canonical macrophage markers. Each of these macrophage populations have defining cell surface markers and unique transcriptional profiles that suggest individual functions (Condeelis and Pollard, 2006; Qian and Pollard, 2010). Metastasis is the major cause of the failure of therapy and therefore death in cancer patients. Therefore we have focused our recent research on this process. We showed that macrophages are rapidly recruited to tumor cells as they metastasize to the lung. Ablation of these metastasis-associated macrophages (MAMs) using either genetic or pharmacological means results in an inhibition of tumor cell seeding and subsequent growth. Importantly ablation of these macrophages after the tumor cells have become established also inhibited subsequent metastatic growth suggesting that these cells represent therapeutic targets. Using ex vivo imaging methods we showed that a major action of these macrophages was on the promotion of tumor cell extravasation with subsequent effects on viability. This effect of macrophage on tumor cells could be recapitulated in an ex vivo extravasation assay thus enabling the interrogation of specific signaling molecules that may have function in promoting extravasation. To analyze the source of these MAMs, we performed adoptive transfers of the two different populations of monocytes that are defined by the presence or absence of the surface antigen Ly6C. Interestingly there was preferential recruitment of the Ly6C+ population to the metastatic sites whether in an experimental model of metastasis or into spontaneous metastases from PyMT tumors. Tracking of these monocytes showed that they rapidly differentiated into MAMs in the lung. A similar monocytic population (CD14+CD16-) circulating in human peripheral blood was recruited to human breast cancer metastases following adoptive transfer into immunocompromised mice. Furthermore, in mice we found that the Ly6C+ monocytes are preferentially recruited to bone metastases. In contrast to these data, Ly6C negative monocytes are recruited to the spontaneous tumors and to liver metastases there is a reduction in the recruitment of both populations of monocytes. Inhibition of the recruitment of these Ly6C+ monocytes inhibited tumor cell extravasation and thus metastases in bone and lung but not liver. These data suggest that alternative and tissue-specific mechanisms are operative at different metastatic sites. The dynamic acquisition of monocytic populations to primary and metastatic tumors and their differentiation into distinct macrophage populations represents the potential to target aspects of the tumor microenvironment. Such novel therapeutics in combination with conventional chemotherapeutic and/or radiation therapy may provide synergistic benefits to the patient. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr PL03-01. doi:10.1158/1538-7445.AM2011-PL03-01

Direct and indirect contribution of bone marrow‐derived cells to cancer

Stromal-epithelial interactions may control the growth and initiation of cancers. Here, we not only test the hypothesis that bone marrow-derived cells may effect development of cancers arising from other tissue cells by forming tumor stroma but also that sarcomas may arise by transformation of stem cells from the bone marrow and epithelial cancers may arise by transdifferentiation of bone marrow stem cells to epithelial cancers. Lethally irradiated female FVB/N mice were restored with bone marrow (BM) transplants from a male transgenic mouse carrying the polyoma middle T-oncoprotein under the control of the mouse mammary tumor virus promoter (MMTV-PyMT) and followed for development of lesions. All of 8 lethally irradiated female FVB/N recipient mice, restored with BM transplants from a male MMTV-PyMT transgenic mouse, developed Y-chromosome negative (Y-) cancers of various organs surrounded by Y+ stroma. One of the female FVB/N recipient mice also developed fibrosarcoma and 1, a diploid breast adenocarcinoma containing Y chromosomes. In contrast, only 1 of 12 control female mice restored with normal male BM developed a tumor (lymphoma) during the same time period. These results indicate not only that the transgenic BM-derived stromal cells may indirectly contribute to development of tumors in recipient mice but also that sarcomas may arise by transformation of BM stem cells and that breast cancers arise by transdifferentiation of BM stem cells, presumably by mesenchymal-epithelial transition.

Synthetic cannabinoid receptor agonists inhibit tumor growth and metastasis of breast cancer

Cannabinoids have been reported to possess antitumorogenic activity. Not much is known, however, about the effects and mechanism of action of synthetic nonpsychotic cannabinoids on breast cancer growth and metastasis. We have shown that the cannabinoid receptors CB1 and CB2 are overexpressed in primary human breast tumors compared with normal breast tissue. We have also observed that the breast cancer cell lines MDA-MB231, MDA-MB231-luc, and MDA-MB468 express CB1 and CB2 receptors. Furthermore, we have shown that the CB2 synthetic agonist JWH-133 and the CB1 and CB2 agonist WIN-55,212-2 inhibit cell proliferation and migration under in vitro conditions. These results were confirmed in vivo in various mouse model systems. Mice treated with JWH-133 or WIN-55,212-2 showed a 40% to 50% reduction in tumor growth and a 65% to 80% reduction in lung metastasis. These effects were reversed by CB1 and CB2 antagonists AM 251 and SR144528, respectively, suggesting involvement of CB1 and CB2 receptors. In addition, the CB2 agonist JWH-133 was shown to delay and reduce mammary gland tumors in the polyoma middle T oncoprotein (PyMT) transgenic mouse model system. Upon further elucidation, we observed that JWH-133 and WIN-55,212-2 mediate the breast tumor-suppressive effects via a coordinated regulation of cyclooxygenase-2/prostaglandin E2 signaling pathways and induction of apoptosis. These results indicate that CB1 and CB2 receptors could be used to develop novel therapeutic strategies against breast cancer growth and metastasis.

Ras- and PI3K-dependent breast tumorigenesis in mice and humans requires focal adhesion kinase signaling

Cancer cells require sustained oncogenic signaling in order to maintain their malignant properties. It is, however, unclear whether they possess other dependencies that can be exploited therapeutically. We report here that in a large fraction of human breast cancers, the gene encoding focal adhesion kinase (FAK), a core component of integrin signaling, was amplified and FAK mRNA was overexpressed. A mammary gland-specific deletion of Fak in mice did not seem to affect normal mammary epithelial cells, and these mice were protected from tumors initiated by the polyoma middle T oncoprotein (PyMT), which activates Ras and PI3K. FAK-deficient PyMT-transformed cells displayed both growth arrest and apoptosis, as well as diminished invasive and metastatic capacity. Upon silencing of Fak, mouse mammary tumor cells transformed by activated Ras became senescent and lost their invasive ability. Further, Neu-transformed cells also underwent growth arrest and apoptosis if integrin beta4-dependent signaling was simultaneously inactivated. Human breast cancer cells carrying oncogenic mutations that activate Ras or PI3K signaling displayed similar responses upon silencing of FAK. Mechanistic studies indicated that FAK sustains tumorigenesis by promoting Src-mediated phosphorylation of p130Cas. These results suggest that FAK supports Ras- and PI3K-dependent mammary tumor initiation, maintenance, and progression to metastasis by orchestrating multiple core cellular functions, including proliferation, survival, and avoidance of senescence.

The Expression Signatures of Mammary Tumor Associated Macrophages.

Abstract It is well established that an increased density of tumor-associated macrophages (TAMs) correlates with poor prognosis in many types of solid tumors. This evidence is particularly strong for breast cancers. A causal relationship between TAMs and poor prognosis was suggested by experiments whereby a genetic depletion of macrophages in a mouse model of breast cancer caused by the mammary restricted expression of the Polyoma Middle T oncoprotein (PyMT) slowed tumor progression and inhibited metastasis. Subsequent studies in these primary mammary tumors showed that TAMs directly or indirectly promote tumor angiogenesis as well as tumor cell migration, invasion and intravasion. TAMs are also thought to affect the inflammatory context of the tumor microenvironment by suppressing adaptive immune responses that would normally reject the growing tumor. The varied tasks ascribed to TAMs suggested that the tumor microenvironment educates different population of macrophages to perform specific tasks. In this study, we isolated TAMs from the PyMT primary tumors in order to evaluate their gene expression signatures compared to a resident splenic population in order to define specific tumor associated functions. To perform these studies mice that express enhanced green fluorescent protein (eGFP) from the colony stimulating factor 1 receptor (CSF-1R) mononuclear phagocytic restricted promoter were crossed to the PyMT animals to generate offspring with eGFP+ TAMs. Animals were injected with dye-conjugated dextran two hours prior to sacrifice in order to identify phagocytic cells, a characteristic of macrophages. We have established that these eGFP+/dextran+ cells are F4/80+ and define the TAM population. EGFP+/dextran+ TAMs were isolated using flow cytometry from late-stage tumors and splenic macrophages were sorted from non-tumor bearing animals using an identical protocol. These two populations were analyzed on gene expression oligoarrays to better elucidate specific mediators of TAM pathogenicity. We have identified approximately 100 genes whose transcript abundance are up or down regulated in the TAM population including genes mediating angiogenesis, adhesion and inflammation. Furthermore, genes previously described to define the tumor associated suppressor macrophage (MIF-1, MIP1α and TGFβ, high; IL-18, low) were similarly regulated amongst the three biological repeats. To further define individual TAM populations, we used an in vivo invasion assay to isolate a subset of TAMs that promote carcinoma cell motility in vivo. This assay involves the collection of invasive tumor cells and co-migrating invasive TAMs into EGF-containing microneedles placed directly into the primary tumor of an MMTV-PyMT animal. Previously, this assay was used to describe a paracrine loop in which carcinoma cells secrete CSF-1 that binds CSF-1R on TAMs leading to TAM secretion of epidermal growth factor (EGF) that binds the EGF receptor on carcinoma cells and stimulate their motility. Disrupting this paracrine loop is known to block the invasion of both cell types. Invasive TAMs isolated via this assay and separated from invasive carcinoma cells using CD11b magnetic beads were compared by gene expression arrays to TAMs sorted by flow cytometry (F4/80+/dextran+). The transcript abundance of about 200 genes were differentially regulated between these two populations. Together, these two studies illustrate key genes expressed in TAMs that may regulate tumor progression and furthermore, define a specific sub-population of TAMs that directly promotes tumor cell migration and invasion.

Vascular endothelial growth factor restores delayed tumor progression in tumors depleted of macrophages

Genetic depletion of macrophages in Polyoma Middle T oncoprotein (PyMT)-induced mammary tumors in mice delayed the angiogenic switch and the progression to malignancy. To determine whether vascular endothelial growth factor A (VEGF-A) produced by tumor-associated macrophages regulated the onset of the angiogenic switch, a genetic approach was used to restore expression of VEGF-A into tumors at the benign stages. This stimulated formation of a high-density vessel network and in macrophage-depleted mice, was followed by accelerated tumor progression. The expression of VEGF-A led to a massive infiltration into the tumor of leukocytes that were mostly macrophages. This study suggests that macrophage-produced VEGF regulates malignant progression through stimulating tumor angiogenesis, leukocytic infiltration and tumor cell invasion.

Treatment with rhenium-188-perrhenate and iodine-131 of NIS-expressing mammary cancer in a mouse model remarkably inhibited tumor growth

Novel therapeutic modalities are needed for breast cancer patients in whom standard treatments are not effective. Mammary gland sodium/iodide symporter has been identified as a molecular target in breast cancers in humans and in some transgenic mouse models. We report the results of a therapy study with (131)I(-) and (188)ReO(4)(-) of breast cancer in polyoma middle T oncoprotein (PyMT) transgenic mice endogenously expressing the Na(+)/I(-) symporter (NIS). PyMT mice (12-13 weeks old) with one palpable tumor of 0.5-0.8 cm in diameter were used. For the therapy studies, PyMT mice were (1) treated with two intraperitoneal injections of 1.5 mCi of (188)ReO(4)(-) 1 week apart, (2) pretreated for 1 week with 5 microg of triiodothyronine (T3) followed by two intraperitoneal injections of 1.5 mCi of (131)I(-) 1 week apart or (3) left untreated. The tumor and normal organ uptakes were assessed by scintigraphic imaging. The thyroid function of treated and control animals was evaluated at the completion of the study by measuring the T3/thyroxine (T4) ratio in their blood. There was significant uptake of (131)I(-) and (188)ReO(4)(-) in the primary palpable tumors as well as in nonpalpable tumors, stomachs and thyroids. The tumor uptake after the second injection was 10 times lower in comparison with the first injection. Tumor growth was significantly inhibited in both the (131)I(-) and (188)ReO(4)(-) groups in comparison with the control group, and tumors in the (188)ReO(4)(-) group increased in size significantly less than in the (131)I(-) group. The T3/T4 ratios were calculated to be 27 and 25 for the control group and the (188)ReO(4)(-) group, respectively; for (131)I(-), both the T3 and T4 levels were below detection limit, demonstrating much less effect on the thyroids of treatment with (188)ReO(4)(-) than with (131)I(-). These results prove that NIS expression in breast tumors in animal models allows specific, efficient and safe treatment with a variety of radionuclides transported by NIS.

Progression to Malignancy in the Polyoma Middle T Oncoprotein Mouse Breast Cancer Model Provides a Reliable Model for Human Diseases

Animal models are powerful tools to analyze the mechanism of the induction of human breast cancer. Here we report a detailed analysis of mammary tumor progression in one mouse model of breast cancer caused by expression of the polyoma middle T oncoprotein (PyMT) in the mammary epithelium, and its comparison to human breast tumors. In PyMT mice, four distinctly identifiable stages of tumor progression from premalignant to malignant stages occur in a single primary tumor focus and this malignant transition is followed by a high frequency of distant metastasis. These stages are comparable to human breast diseases classified as benign or in situ proliferative lesions to invasive carcinomas. In addition to the morphological similarities with human breast cancer, the expression of biomarkers in PyMT-induced tumors is also consistent with those associated with poor outcome in humans. These include a loss of estrogen and progesterone receptors as well as integrin-beta1 expression and the persistent expression of ErbB2/Neu and cyclinD1 in PyMT-induced tumors as they progress to the malignant stage. An increased leukocytic infiltration was also closely associated with the malignant transition. This study demonstrates that the PyMT mouse model is an excellent one to understand the biology of tumor progression in humans.