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In previous studies (Chen, W. Y. et al., Clin. Cancer Res., 5:3583-3593, 1999; Chen, N Y. et al., Int. J. Oncol., 20:813-818, 2002), we have demonstrated the ability of the human prolactin (hPRL) antagonist, G129R, to inhibit human breast cancer cell proliferation in vitro and to slow the growth rate of tumors in mice. We further revealed that the possible mechanisms of G129R antitumor effects act through the induction of apoptosis via the regulation of bcl-2 gene expression. It has been established that to sustain tumor growth, it is necessary for the development of a network of blood vessels to bring in nutrients, a process called angiogenesis. The disruption of angiogenesis has been proven to be an effective strategy to cause regression of certain tumors. One of the best-studied angiogenesis inhibitors is endostatin, which acts through the inhibition of endothelial cells. In this study, we combine the anti-breast tumor effects of G129R and the antiangiogenic effects of endostatin by creating a novel fusion protein (G129R-endostatin) specifically for breast cancer therapy. The data presented here demonstrated that this novel fusion protein was able to bind to the PRL receptor (PRLR) on T-47D human breast cancer cells and inhibit the signal transduction induced by PRL. At the same time, G129R-endostatin inhibited human umbilical vein endothelial cell (HUVEC) proliferation and disrupted the formation of endothelial tube structures with potency similar to that of endostatin. More importantly, the therapeutic efficacy of G129R-endostatin was confirmed using a mouse breast cancer cell line 4T1 in vivo. G129R-endostatin has a significantly prolonged serum half-life as compared with that of G129R or endostatin alone, and exhibited greater tumor inhibitory effects than G129R and endostatin individually or in combination. Taken together, these data demonstrate the dual therapeutic effects of G129R-endostatin, and suggests that this fusion protein has great promise as a novel anti-breast cancer agent.

Adenylyl cyclase (AC) catalyzes the conversion of ATP to the second messenger cAMP, which in turn activates protein kinase A (PKA). AC/cAMP/PKA signaling pathway is known to mediate tumor growth but the results of previous studies have been controversial. More importantly, little is known about how specific AC isoforms affect tumor growth. Based on our previous data showing that knocking out the isoform AC type 5 in mice (AC5KO) extends longevity and that the major cause of premature mortality in mice is neoplastic diseases, we hypothesized that AC5KO mice can be protected against tumor growth. We previously demonstrated that AC5KO mice are protected against oxidative stress and obesity, which should also provide protection against neoplastic disease. In order to elucidate whether AC5 plays a role in the tumor microenvironment, we initially investigated the growth of B16F10 melanoma in this model. We have found that AC5KO mice exhibit protection against B16F10 melanoma tumor growth (p Tumor latency, wet tumor weights and tumor volume were also significantly reduced in AC5KO mice (p Obesity is a risk factor for the development of breast cancer. In accord with the fact that AC5KO are protected against obesity, we have found that circulating levels of leptin were significantly reduced in AC5KO: 1.99±0.28 vs. WT: 3.23±0.35 ng/ml (p Citation Format: Mariana S. De Lorenzo, Wen Y. Chen, Erdene Baljinnyam, Krista La Perle, Sanford P. Bishop, Thomas E. Wagner, Patricio Abarzua, Arnold B. Rabson, Dorothy E. Vatner, Lydia I. Puricelli, Stephen F. Vatner. Protection against tumor growth by disruption of adenylyl cyclase type 5 (AC5). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-337. doi:10.1158/1538-7445.AM2013-LB-337

Background: Prolactin (PRL), a polypeptide hormone, has been implicated in breast tumorigenesis via promoting cell proliferation and survival. One of the mechanisms by which PRL is linked to breast cancer is through cross-talk with other oncogenic growth factors including human epidermal growth factor receptor 2 (HER2/neu). Studies have demonstrated that PRL is able to cross-activate HER2/neu and that a PRL receptor (PRLR) antagonist, G129R, is able to inhibit HER2/neu phosphorylation (pHER2/neu) in established breast cancer cell lines. In this study, we investigate the role of tumor associated fibroblasts (stromal cells) in this molecular cross-talk process, using natural tumors and tumor epithelial and fibroblast cell lines derived from female MMTV/neu transgenic mice. Methods: (1) Primary tumor studies. Mammary tumors from MMTV/neu transgenic mice were surgically removed and divided into two portions. One portion was cut into small chunks (3 mm3 cubes) and cultured three-dimensionally. The other portion was minced and digested into a single cell suspension and cultured as a monolayer. (2) Direct co-culture of tumor epithelial and fibroblasts studies. A tumor epithelial cell line, McNeuA, and tumor associated fibroblast cell line, N202Fb3, derived from a MMTV/neu mammary tumor (kindly provided by Dr Campbell, UC San Francisco) were cultured alone or mix-cultured at different ratios. A normal mouse fibroblast cell line was used as control. (3) Transwell indirect co-culture studies. McNeuA and N202Fb3 were co-cultured indirectly using a transwell system to allow exposure to soluble factors secreted by both cell types. Tumor cells in all three experimental conditions were treated with various concentrations of either PRL or G129R for 1-, 6-, 24-, or 48 hours. The effects of the treatments on pHER2/neu and downstream signaling molecules were evaluated by immunoblotting. Results: (1) G129R inhibited the pHER2/neu in a dose-dependent manner (IC50=10μg/ml) after 24hr treatment in the tumor chunks (three-dimensional model), but had no effect in the monolayer primary culture; (2) Direct co-culture of McNeuA and N202Fb3 restored the response of McNeuA to G129R similar to that of tumor chunks with a best result at 4 (McNeuA) : 1 (N202Fb3) ratio. The inhibitory effect of G129R was reversed by addition of PRL. Furthermore, the inhibitory effect of G129R in McNeuA was vanished when N202Fb3 was replaced with normal fibroblasts. (3) Similarly, G129R had no effect upon McNeuA when N202Fb3 were separated in the transwell co-culture system. Conclusions: In this study we have demonstrated that the tumor stromal cells, in particular, the tumor associated fibroblasts, play an important role in bridging the cross-talk between PRLR and HER2/neu. The inhibitory effect of G129R on pHER2/neu in tumor epithelial cells is dependent upon physical contact with tumor associated fibroblasts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1007.

Background. Tumors are organized in a cellular hierarchy, in which cancer stem cells (CSCs) are the only cells that have the ability of self-renewal and differentiation. CSCs generally are in quiescent state and resistant to chemotherapy, which is one of the reasons that using conventional chemotherapeutics often leads to enrichment in CSCs. It is known that adult stem cells (ASCs) and CSCs share many common features. In adult mammary tissue, one of the distinctive features is its ability to go through multiple proliferation and involution cycles during pregnancy, suggesting that ASCs in this organ are readily responsive to female hormone regulations. One such hormone that plays a crucial role in this process is human prolactin (hPRL), which is required for mammary lobuloalveolar formation, terminal differentiation, and for synthesis of milk components. In this study, we hypothesize that simultaneous administration of hPRL with chemotherapeutic agents such as doxorubicin (Dox) will increase the cytotoxic effects of the chemotherapeutic drugs towards CSCs through the induction of CSCs proliferation/differentiation by hPRL. Methods. MCF-7 human breast cancer cells and primary mouse breast cancer cells isolated from MMTV-neu transgenic mice were used in this study. Tumorsphere formation assay and ALDH activity measured by flow cytometry were used to measure the functional response of the CSCs after co-treatment of hPRL and Dox. Results. (1) hPRL treatment increased tumorspheres formation in both MCF-7 (1.3-fold ± 0.24, p Conclusion. Our preliminary results suggest that hPRL is able to improve Dox's efficacy towards CSCs. Further studies are needed to validate the results. This novel therapeutic approach may open doors for designing new strategy for breast cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3329.