Biological responses to TGF-ß in the mammary epithelium show a complex dependency on Smad3 gene dosage with important implications for tumor progression.

TGF-ß plays a dual role in epithelial carcinogenesis with the potential to either suppress or promote tumor progression. We found that levels of Smad3 mRNA, a critical mediator of TGF-ß signaling, are reduced by approximately 60% in human breast cancer. We therefore used conditionally immortalized mammary epithelial cells (IMEC) of differing Smad3 genotypes to quantitatively address the Smad3 requirement for different biologic responses to TGF-ß. We found that a two-fold reduction in Smad3 gene dosage led to complex effects on TGF-ß responses; the growth-inhibitory response was retained, the pro-apoptotic response was lost, the migratory response was reduced, and the invasion response was enhanced. Loss of the pro-apoptotic response in the Smad3(+/-) IMECs correlated with loss of Smad3 binding to the Bcl-2 locus, whereas retention of the growth-inhibitory response in Smad3 IMECs correlated with retention of Smad3 binding to the c-Myc locus. Addressing the integrated outcome of these changes in vivo, we showed that reduced Smad3 levels enhanced metastasis in two independent models of metastatic breast cancer. Our results suggest that different biologic responses to TGF-ß in the mammary epithelium are differentially affected by Smad3 dosage and that a mere two-fold reduction in Smad3 is sufficient to promote metastasis.

A novel approach for the generation of genetically modified mammary epithelial cell cultures yields new insights into TGFß signaling in the mammary gland.

INTRODUCTION: Molecular dissection of the signaling pathways that underlie complex biological responses in the mammary epithelium is limited by the difficulty of propagating large numbers of mouse mammary epithelial cells, and by the inability of ribonucleic acid interference (RNAi)-based knockdown approaches to fully ablate gene function. Here we describe a method for the generation of conditionally immortalized mammary epithelial cells with defined genetic defects, and we show how such cells can be used to investigate complex signal transduction processes using the transforming growth factor beta (TGFß/Smad pathway as an example. METHODS: We intercrossed the previously described H-2Kb-tsA58 transgenic mouse (Immortomouse) which expresses a temperature-sensitive mutant of the simian virus-40 large T-antigen (tsTAg), with mice of differing Smad genotypes. A panel of conditionally immortalized mammary epithelial cell (IMEC) cultures were derived from the virgin mammary glands of offspring of these crosses and used to assess the Smad dependency of different biological responses to TGFß. RESULTS: IMECs could be propagated indefinitely at permissive temperatures and had a stable epithelial phenotype, resembling primary mammary epithelial cells with respect to several criteria, including responsiveness to TGFß. Using this panel of cells, we demonstrated that Smad3, but not Smad2, is necessary for TGFß-induced apoptotic, growth inhibitory and EMT responses, whereas either Smad can support TGFß-induced invasion as long as a threshold level of total Smad is exceeded. CONCLUSIONS: This work demonstrates the practicality and utility of generating conditionally immortalized mammary epithelial cell lines from genetically modified Immortomice for detailed investigation of complex signaling pathways in the mammary epithelium.

Ras activation contributes to the maintenance and expansion of Sca-1pos cells in a mouse model of breast cancer.

The cancer stem cell (CSC) hypothesis proposes that CSCs are the root of cancer and cause cancer metastasis and recurrence. In this study, we examined whether Ras signaling is associated with stemness of the CSCs population characterized by the stem cell antigen (Sca-1) phenotype in a 4T1 syngeneic mouse model of breast cancer. The Sca-1(pos) putative CSCs had high levels of activated Ras and phosphorylated MEK (p-MEK), compared with counterparts. The Ras farnesylation inhibitor (FTI-277) suppressed the maintenance and expansion of CSCs. Therefore, selective inhibition of Ras activation may be useful for stem-specific cancer therapy.

Chemokine (C-C motif) ligand 2 mediates the prometastatic effect of dysadherin in human breast cancer cells.

Dysadherin, a cancer-associated membrane glycoprotein, down-regulates E-cadherin and promotes cancer metastasis. This study examined the role of dysadherin in breast cancer progression. Expression of dysadherin was found to be highest in breast cancer cell lines and tumors that lacked the estrogen receptor (ER). Knockdown of dysadherin caused increased association of E-cadherin with the actin cytoskeleton in breast cancer cell lines that expressed E-cadherin. However, knockdown of dysadherin could still suppress cell invasiveness in cells that had no functional E-cadherin, suggesting the existence of a novel mechanism of action. Global gene expression analysis identified chemokine (C-C motif) ligand 2 (CCL2) as the transcript most affected by dysadherin knockdown in MDA-MB-231 cells, and dysadherin was shown to regulate CCL2 expression in part through activation of the nuclear factor-kappaB pathway. The ability of dysadherin to promote tumor cell invasion in vitro was dependent on the establishment of a CCL2 autocrine loop, and CCL2 secreted by dysadherin-positive tumor cells also promoted endothelial cell migration in a paracrine fashion. Finally, experimental suppression of CCL2 in MDA-MB-231 cells reduced their ability to metastasize in vivo. This study shows that dysadherin has prometastatic effects that are independent of E-cadherin expression and that CCL2 could play an important role in mediating the prometastatic effect of dysadherin in ER-negative breast cancer.

Distinct roles for p53 transactivation and repression in preventing UCN-01-mediated abrogation of DNA damage-induced arrest at S and G2 cell cycle checkpoints.

The topoisomerase I inhibitor SN38 arrests cell cycle progression primarily in S or G(2) phases of the cell cycle in a p53-independent manner. The Chk1 inhibitor, 7-hydroxystaurosporine (UCN-01), overcomes both S and G(2) arrest preferentially in cells mutated for p53, driving cells through a lethal mitosis and thereby enhancing cytotoxicity. The mechanism by which p53 maintains S and G(2) arrest was investigated here. The p53 wild-type MCF10A cells were arrested in S phase by incubation with SN38 for 24 h. Subsequent incubation with UCN-01 failed to abrogate arrest. To examine the impact of p53, MCF10A cells were developed, which express the tetramerization domain of p53 to inhibit endogenous p53 function. These cells were attenuated in SN38-mediated induction of p21(WAF1), and UCN-01 induced S, but not G(2) progression. In contrast, MCF10A cells expressing short hairpin RNA to ablate p53 expression underwent both S and G(2) phase progression with UCN-01. The difference in G(2) progression was attributed to p53-mediated gene repression; the MCF10A cells expressing the tetramerization domain retained p53 protein and repressed both cyclin B and Chk1, while cells ablated for p53 did not repress these proteins. Hence, inhibition of p53 activator function permits S phase abrogation, while additional inhibition of p53 repressor function is required for abrogation of G(2) arrest. These studies provide a mechanistic explanation for how this therapeutic strategy can selectively target tumor cells.

The protein kinase C inhibitor Gö6976 is a potent inhibitor of DNA damage-induced S and G2 cell cycle checkpoints.

In response to DNA damage, cells arrest progression through the cell cycle at either G(1), S, or G(2). We have reported that UCN-01 (7-hydroxystaurosporine) abrogates DNA damage-induced S and G(2) arrest and enhances cytotoxicity selectively in p53 mutant cells, thus providing a potential, tumor-targeted therapy. Unfortunately, UCN-01 binds avidly to human plasma proteins, limiting bioavailability. Because UCN-01 also inhibits protein kinase C (PKC), we screened other PKC inhibitors, expecting them to be unable to abrogate arrest. However, Gö6976 potently abrogated S and G(2) arrest and enhanced the cytotoxicity of the topoisomerase I inhibitor SN38 only in p53-defective cells. Importantly, Gö6976 was nearly as potent at abrogating S and G(2) arrest in human serum, a property not possessed by UCN-01. Cell viability studies demonstrated that Gö6976 was impressively nontoxic as a single agent. Analysis of proteins that regulate cell cycle arrest suggested that both drugs inhibit the checkpoint kinases Chk1 and/or Chk2. Additionally, Gö6976 abrogated S and G(2) arrest at a concentration substantially lower than that required to inhibit PKC; UCN-01 did not demonstrate this selectivity for checkpoint inhibition. These properties make Gö6976 a promising candidate for preclinical and clinical studies.

A novel indolocarbazole, ICP-1, abrogates DNA damage-induced cell cycle arrest and enhances cytotoxicity: similarities and differences to the cell cycle checkpoint abrogator UCN-01.

DNA damaging agents such as cisplatin arrest cell cycle progression at either G1, S, or G2 phase, although the G1 arrest is only seen in cells expressing the wild-type p53 tumor suppressor protein. We have reported that 7-hydroxystaurosporine (UCN-01) overcomes S and G2 phase arrest and enhances the cytotoxicity of cisplatin. Abrogation of arrest appears to be selective for cells defective in p53 and therefore provides a potential, tumor-targeted therapy. Unfortunately, UCN-01 binds avidly to human plasma proteins, limiting access to the tumor. A screen of related indolocarbazoles identified analogues with both beneficial and undesirable properties. This led to a synthetic program to develop a novel analogue rationally designed to overcome the obstacles observed with the other analogues. We report the synthesis and analysis of a novel analogue, ICP-1. This analogue abrogated S and G2 phase arrest and enhanced cytotoxicity induced by cisplatin only in p53 defective cells. ICP-1 also abrogated arrest and enhanced cell killing induced by the topoisomerase I inhibitor SN38. Analysis of proteins that regulate cell cycle arrest suggest both drugs inhibit checkpoint kinases Chk1 and/or Chk2. In contrast to UCN-01, checkpoint abrogation by ICP-1 was only slightly inhibited by human plasma. UCN-01 and ICP-1 differed significantly in other regards. UCN-01 potently enhanced the activity of 1-beta-D-arabinofuranosylcytosine in both p53 wild-type and mutant cells, whereas ICP-1 was inactive in this combination. This property of UCN-01 was independent of its ability to inhibit protein kinase C because more specific inhibitors of protein kinase C failed to enhance cell killing induced by 1-beta-D-arabinofuranosylcytosine. High concentrations of UCN-01 also inhibit C-TAK1 that results in S phase-arrested cells directly entering mitosis, but this property was not observed with ICP-1. Hence, ICP-1 appears to be a more selective inhibitor of the S and G2 cell cycle checkpoint than previously studied analogues and is worthy of study in preclinical tumor models.

Abrogation of the S phase DNA damage checkpoint results in S phase progression or premature mitosis depending on the concentration of 7-hydroxystaurosporine and the kinetics of Cdc25C activation.

DNA damage causes cell cycle arrest in G(1), S, or G(2) to prevent replication on damaged DNA or to prevent aberrant mitosis. The G(1) arrest requires the p53 tumor suppressor, yet the topoisomerase I inhibitor SN38 induces p53 after the G(1) checkpoint such that the cells only arrest in S or G(2). Hence, SN38 facilitates comparison of p53 wild-type and mutant cells with regard to the efficacy of drugs such as 7-hydroxystaurosporine (UCN-01) that abrogate S and G(2) arrest. UCN-01 abrogated S and G(2) arrest in the p53 mutant breast tumor cell line MDA-MB-231 but not in the p53 wild-type breast line, MCF10a. This resistance to UCN-01 in the p53 wild-type cells correlated with suppression of cyclins A and B. In the p53 mutant cells, low concentrations of UCN-01 caused S phase cells to progress to G(2) before undergoing mitosis and death, whereas high concentrations caused rapid premature mitosis and death of S phase cells. UCN-01 inhibits Chk1/2, which should activate the mitosis-inducing phosphatase Cdc25C, yet this phosphatase remained inactive during S phase progression induced by low concentrations of UCN-01, probably because Cdc25C is also inhibited by the constitutive kinase, C-TAK1. High concentrations of UCN-01 caused rapid activation of Cdc25C, which is attributed to inhibition of C-TAK1, as well as Chk1/2. Hence, UCN-01 has multiple effects depending on concentration and cell phenotype that must be considered when investigating mechanisms of checkpoint regulation.