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Several molecules that suppress metastasis without suppressing tumorigenicity have been identified, but their mechanisms of action have not yet been determined. Many block growth at the secondary site, suggesting involvement in how cells respond to signals from the extracellular milieu. Breast cancer metastasis suppressor 1 (BRMS1)–transfected MDA-MB-435 cells were examined for modifications of phosphoinositide signaling as a potential mechanism for metastasis suppression. 435/BRMS1 cells expressed

Several molecules that suppress metastasis without suppressing tumorigenicity have been identified, but their mechanisms of action have not yet been determined. Many block growth at the secondary site, suggesting involvement in how cells respond to signals from the extracellular milieu. Breast cancer metastasis suppressor 1 (BRMS1)-transfected MDA-MB-435 cells were examined for modifications of phosphoinositide signaling as a potential mechanism for metastasis suppression. 435/BRMS1 cells expressed10% of phosphatidylinositol-4, 5-bisphosphate compared with parental cells, whereas levels of the PtdIns(4)P and phosphatidylinositol-3-phosphate were unchanged. Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] were decreased in 435/BRMS1 cells by approximately 50%. Phosphatidylinositol-3,4,5-trisphosphate levels were undetectable in 435/BRMS1 cells, even when stimulated by exogenous insulin or platelet-derived growth factor. Immunofluorescence microscopy to examine cellular distribution confirmed that phosphatidylinositol-4,5-bisphosphate distribution with cells was unchanged but was uniformly decreased throughout the cell. Although the gross morphology of 435/BRMS1 cells is similar to the parent, filamentous actin was more readily apparent in 435/BRMS1. Intracellular calcium, measured using Fluo-3 and Fura-2 fluorescent calcium indicator dyes, was somewhat lower, but not statistically different in 435/BRMS1 compared with parental cell. However, when stimulated with platelet-derived growth factor, MDA-MB-435 cells, but not 435/BRMS1 cells mobilized intracellular calcium. Taken together, these results implicate signaling through phosphoinositides in the regulation of breast cancer metastasis, specifically metastasis that can be suppressed by BRMS1.

Cancer cells show epithelial-mesenchymal transition (EMT) during cell migration, invasion and dissemination. Although it has been shown by numerous publications that the evolutionary conserved basic helix-loop-helix (b-HLH) transcription factor TWIST1 plays pivotal role during EMT the molecular mechanism responsible for TWIST1 function is not fully understood. Here we show that Twist1 binds to and phosphorylated by AKT/protein kinase-B at S42, T121 and S123. While conversion of S42, T121 and S123 to phosphorylation-mimicking Aspartic or glutamic acids created active Twist1, Alanin mutants of the same sites diminished the DNA-binding and transctivating functions of Twist1. In line with this, Glutamic Acids mutants suppressed the expression of E-Cadherin, whose expression is negatively regulated by active TWIST1, Alanin mutants induced the expression of E-Cadherin. Similarly, we tested the impact of above mentioned mutants on cell migration and proliferation. Our results demonstrated that while Glutamic acid mutants accelerated, Alanin mutants suppressed the migration and proliferation of 293T cells. Our in vitro results prompted us to test our mutants under in vivo conditions using breast cancer as a model. We transfected non-metastatic mouse mammary tumor cell line, 67NR, with Glutamic acid mutants, and metastatic mouse mammary tumor cell line, 4T1, with Alanin mutants of mouse TWIST1 and selected the Neo-resistant populations. We injected 1 million 67NR cells over-expressing wild type or Glutamic acid mutants of Twist1, and 100.000 4T1 cells over-expressing wild type or Alanin mutants of Twist1 into the breast tissue of female inbred BalBC mice, and animals were monitored for metastasis. At the end of the 4th week, we sacrifised and examined the animals for tumor growth and metastasis. Although 67NR cells over-expressing wild type Twist1 did not show any metastasis, cells over-expressing 1E (S42E) and 2E (S42E, T121E) mutants of Twist1 showed 15-20 macroscopic metastasis to liver and Lungs. Paralel to this, 4T1 cells expressing 1A (S42A) and 2A (S42A, T121A) mutants of TWIST1 showed no macroscopic metastasis. Our results clearly indicate that phosphorylation of S42 and T121 by AKT is essential for TWIST1-mediated tumor growth and metastasis. Citation Format: Osman N. Ozes, Suray Pehivanoglu, Gokhan Ertosun, Gokhan Gorgisen, Nuray Erin, Duygu Unal, Gamze Tanriover. AKT-mediated phosphorylation is responsible for TWIST1-mediated tumor growth and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1130. doi:10.1158/1538-7445.AM2014-1130

Carcinomas are formed by heterogeneous group of cells which differ in their aggressiveness as well as metastatic potential. Understanding the characteristics of cells that form distant metastasis is crucial to design effective treatments. We previously isolated liver metastatic cells (4TLM) formed by 4THMpc murine breast carcinoma using an orthotopic model. Here we observed that 4THMpc cells also generated macroscopic brain metastasis. Cells from brain metastasis were cultured in-vitro and named as 4THM-BM in order to determine differential characteristics of brain metastatic cells from liver metastatic cells. Specifically following features were compared: 1. Metastatic characteristics after mammary pad (MP) injection; 2. The effects of conditioned medium (CM) prepared from ex-plants of primary tumors as well as cancer-associated fibroblasts (CAF) on tumor cell growth. 3. MIP-2 levels found in CM of ex-plants of primary tumors and associated fibroblasts. Methods: 4TLM and 4THM-BM cells (100000 cells /mouse) were inoculated into the right upper MP of 8-10 weeks old female Balb-c mice. Necropsies were performed 25-27 days after injection. CM of ex-plants from primary tumors and surrounding fibroblasts were prepared. Results: Cells that were previously metastasized to liver (4TLM) produced significantly more macroscopic (52.2± 8.5 vs. 16.3 ±2.84 as mean number of nodules per animal) as well as microscopic lung metastasis (12.75 ±4.26 vs. 3.17±1,1 as mean % area involved with metastatic cells per animal) compared to brain metastatic cells (4THM-BM). Similarly number of macroscopic liver metastases was significantly higher (3.4± 0,6 vs. 7,3 ±1,23 as mean number of nodules per animal) in 4TLM injected animals. CM from ex-plants of 4TLM and 4THM-BM primary tumors as well as from corresponding CAF significantly enhanced growth of 4TLM cells but not 4THM-BM cells. MIP-2 levels in CM from ex-plants of CAF were higher from CM of 4TLM and 4THM-BM primary tumors. These results not only demonstrate the heterogeneous nature of the carcinomas but also suggest that cells that metastasize to liver are more aggressive in nature and produce more visceral metastasis. MIP-2, murine equivalent to human IL-8, is involved in angiogenesis as well as inflammation and reported to be highly expressed in metastatic cells. Here we found that both primary tumors formed by metastatic cell lines and associated fibroblasts produce MIP-2 which may contribute to the metastatic behavior. Study was supported by The Scientific and Technological Research Council of Turkey (TÜBITAK Grant no:109S449). 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 2426. doi:10.1158/1538-7445.AM2011-2426