Breast cancer (BCa) is the most common malignant disease in women in the U.S. Nearly 20% of patients with advanced BCa are eventually diagnosed with brain lesions, which is a devastating complication in patients with BCa over-expressing EGF receptor family members including Her2 positive and triple negative breast cancer. It is the most feared complication of BCa in part because are not capable of significantly treating the BCa-induced brain metastases due to the inability of the available treatment regimens to effectively penetrate the blood brain barrier (BBB) and also due to our limited knowledge on cellular and molecular mechanisms that drive the homing to and growth in the brain of BCa cells. Therefore, there is a need of efficient model system that can significantly contribute towards our understanding of different factors from both host and tumor leading to brain metastasis. We have recently isolated a novel BCa cell line B6TC that was generated through fusion between human BCa, MDA-MB-231 and ZR-75-1 cells in mouse bone marrow. This B6TC cell line showed higher propensity to metastasize to brain than its parental cells when inoculated through intracardiac injection in female athymic nude mice. In order to generate a highly brain metastatic breast cancer model for mechanistic research, we subjected the B6TC cells through four rounds of selection for cells that were capable of trans-endothelial cell invasion to obtain cells that could invade through BBB. This in vitro selected cell line was further subjected through three rounds of in vivo selection for cells that were capable of metastasizing to the brain and the cells after third round selection was named N3LR, which has the highest potential to cause brain metastasis. In searching for genes and pathways that may contribute to the increased brain metastasis of N3-LR cell with microarray analysis, we found that the transforming growth factor-beta (TGFβ) signaling pathway is upregulated in N3-LR cell in comparison with B6TC cell, in addition to the EGF and prostaglandin signaling pathways that have been reported to be associated with brain metastatic breast cancer cells. Functional comparison also showed that N3-LR cell was more migratory than B6TC cell and more responsive to TGFβ-induced phosphorylation of Smad3 as well as migration, suggesting that TGFβ signaling may contribute to the increased brain metastatic potential. We next investigated whether metastatic tumor growth in the brain microenvironment can be inhibited by systemic administration of a potent pan-TGFβ inhibitor, BGERII- a recombinant fusion protein containing the endoglin domain of betaglycan (BGE) and the extracellular domain of RII. The animals were inoculated intracardically with N3LR, the most potent subline of highly metastatic B6TC cells, and were then treated with vehicle or BGERII systemically via i.p. injection right after the inoculation. After three weeks, the BGERII treated group showed lower brain metastasis incidence and tumor burden as detected by whole mouse bioluminescence and GFP imaging. Further analyses to understand the underlying molecular and regulatory mechanism of brain metastasis and its intervention in our mouse model is underway for the discovery of novel molecularly targeted drugs to prevent and eradicate BCa metastasis initiation, progression and recurrence. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-18.
Breast cancer (BCa) is the most common malignant disease in women in U.S. and the mortality in BCa patients is due to the metastasis of the disease from primary site. Brain metastasis accounts for nearly 20% of all metastases in BCa. It is the most feared complication of BCa because of very few effective treatment regimens available, leading to lower survival rate in patients. The exact molecular mechanism for metastases of BCa into brain is unknown. Rodent model systems have been reported for brain metastasis in BCa but the current models for brain metastasis have limitations. Therefore, there is a need of efficient model system that can significantly contribute towards our understanding of different factors from both host and tumor leading to brain metastasis. Previously we reported isolation and characterization of estrogen independent B6TC cells that are derived from the stable spontaneous fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in mouse bone marrow microenvironment. This chimeric B6TC has propensity to metastasize to brain when inoculated through intracardiac (I.C.) route, and express stem cell-like features. In the present study using B6TC, we have developed an efficient and novel mouse model for studying BCa-induced brain metastasis. We have generated three cell lines from B6TC through three successive rounds of inoculation in mouse and subsequent isolation of brain metastatic cells. Each round of selection enhanced the brain metastatic propensity. An initial microarray analysis identified genes like MMP1, HB-EGF, ST3GAL1, PTGS2, ITGA3, and CXCR4, which showed significant up-regulation in B6TC compared to its parental metastatic MDA-MB-231 or nonaggressive ZR-75-1 cells. These genes are implicated in metastasis regulation. A second round of RNA microarray was performed with three sublines of B6TC with successively enhanced brain metastatic propensity to identify unique potential brain metastatic genes showing gradual up or down-regulation over generations. From analysis of the gene expression profiles, apart from potent brain metastatic genes detected earlier, we also identified some molecular pathways, including TGF beta signaling pathway that are associated with enhanced brain metastasis. The B6TC model is novel for studying the molecular mechanism of brain metastasis, as in this model, apart from experimental metastasis in brain through I.C route, cells show spontaneous metastases to the brain from the primary tumor and this unique feature will enable us to study the mechanisms of the early steps of brain metastasis progression. Further analyses to find out common miRNAs that are over or under-expressed in the sublines over successive generations and determination of gene targets of miRNA are underway. This study will not only provide valuable insight into molecular mechanism of BCa-induced brain metastasis but also lay the foundation to identify novel prognostic and therapeutic markers of brain metastases, leading ultimately to the discovery of novel molecularly targeted drugs to prevent and eradicate BCa metastasis initiation, progression and recurrence. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-12.
Triple negative, basal-like breast cancer is highly aggressive and has a poor prognosis. The molecular mechanisms that drive its progression are elusive and no molecular target has been identified for its prevention or treatment. Here, we demonstrate that triple negative human breast cancer cells and clinical samples show an attenuated transforming growth factor-beta (TGF-β) signaling. Therefore, we developed a series of isogenic basal-like human mammary epithelial cells (HMECs) with altered TGF-β sensitivity and different malignancy, resembling a full spectrum of basal-like breast carcinogenesis, and determined the molecular mechanisms that contribute to oncogene-induced transformation of basal-like HMECs when TGF-β signaling is attenuated. We found that expression of a dominant-negative RII (DNRII) receptor of TGF-β abrogated autocrine TGF-β signaling in telomerase-immortalized HMECs and suppressed H-ras-V12-induced senescence-like growth arrest (SLGA). Furthermore, co-expression of DNRII and H-ras-V12 rendered HMECs to become highly tumorigenic and metastatic in vivo in comparison with H-ras-V12-transformed HMECs that spontaneously escaped H-ras-V12-induced SLGA. By using microarray analysis, we found that p21 is the major player mediating Ras-induce SLGA and attenuated or loss of p21 expression contributed to the escape from SLGA when autocrine TGF-β signaling is blocked in HMECs. Furthermore, knockdown of p21 also suppressed H-ras-V12-induced SLGA. Our results identifies that autocrine TGF-β signaling is an integral part of cellular antitransformation network by suppressing the expression of a host of genes including p21-regulated genes that mediate oncogene-induced transformation in basal-like breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-03-05.
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