BACKGROUND:TBX3 is a T-box transcription factor repressor that is elevated in metastatic breast cancer and is believed to promote malignancy of tumor cells, possibly by promoting cell survival and epithelial-mesenchymal transition.METHODS:The relative expression of TBX3 was assessed in the 21T cell lines which were derived from an individual patient and represent three distinct stages of breast cancer progression: 21PT, atypical ductal hyperplasia; 21NT, ductal carcinoma in situ; and 21MT-1, invasive mammary carcinoma. Two different isoforms of TBX3 (TBX3iso1 and TBX3iso2) were overexpressed to evaluate cell survival/colony forming ability, growth, and invasion in the ductal carcinoma in situ-like 21NT cell line using an in vitro Matrigel model of cancer progression. In addition, TBX3 expression was knocked down to evaluate the effects of downregulating TBX3 on the invasive mammary carcinoma-like 21MT-1 cell line. Finally, PCR array profiling was used to assess alterations in gene expression due to TBX3 overexpression in the 21NT cells.RESULTS:TBX3 is abundant in the invasive 21MT-1 cell line, while being minimally expressed in the non-invasive 21NT and 21PT cell lines. Overexpression of either TBX3iso1 or TBX3iso2 in 21NT cells resulted in increased cell survival/colony forming ability, growth vs. apoptosis and invasion in Matrigel. In contrast, short hairpin RNA-mediated knockdown of TBX3 in the 21MT-1 cells resulted in smaller colonies, with a more regular, less dispersed (less infiltrative) morphology. Array profiling of the 21NT TBX3 iso1 and iso2 transfectants showed that there are common alterations in expression of several genes involved in signal transduction, cell cycle control/cell survival, epithelial-mesenchymal transition and invasiveness.CONCLUSIONS:Overall, these results indicate that TBX3 (isoform 1 or 2) expression can promote progression in a model of early breast cancer by altering cell properties involved in cell survival/colony formation and invasiveness, as well as key regulatory and EMT/invasiveness-related gene expressions.

Planar cell polarity (PCP) signaling has been shown in different studies to either promote or inhibit the malignancy of breast cancer. Using the 21T cell lines, which were derived from an individual patient and represent distinct stages of progression, we show that the prototypical PCP ligand, WNT5A, is expressed highest in 21MT-1 cells (invasive mammary carcinoma) and lowest in 21PT (atypical ductal hyperplasia) and 21NT (ductal carcinoma in situ) cells. Overexpression of WNT5A decreased spherical colony formation and increased invasion and in vivo extravasation only in 21NT cells; whereas overexpression increased migration of both 21PT and 21NT cells. WNT5A overexpression also increased RHOA expression of both cell lines and subsequent RHOA knockdown blocked WNT5A-induced migration, but only partially blocked WNT5A-induced invasion of 21NT cells. PCP can signal through VANGL1 to modulate AP-1 target genes (e.g. MMP3) and induce invasion. VANGL1 knockdown inhibited WNT5A-induced invasion of 21NT cells, but had no effect on WNT5A-induced migration of either 21PT or 21NT cells. WNT5A-induced MMP3 expression was seen only in 21NT cells, an effect that was VANGL1 dependent, but independent of AP-1. We thus provide evidence that PCP signaling can act in a context dependent manner to promote breast cancer progression.

Abstract The transcriptional regulator TBX3 has been shown to be elevated in metastatic breast cancer and is thought to be involved in promoting malignancy of breast and other carcinomas. In a series of cell lines derived from the same patient with breast cancer, we previously showed that metastatic 21MT-1 cells express elevated levels of TBX3, while low levels are expressed by the ductal carcinoma in situ-like 21NT cells. Here we hypothesized that upregulation of TBX3 in 21NT cells would promote their progression to a more malignant phenotype. We found that 21NT cells engineered to overexpress either isoform of TBX3 (iso1, iso2) have an altered growth in 3D Matrigel, with increased colony-forming ability, higher number of cells per colony and less spherical, more irregular colonies, consistent with increased invasiveness. Transwell assays through Matrigel showed increased invasiveness of 21NT cells overexpressing either TBX3 isoform and in vivo assay in the chick embryo similarly showed increased propensity of TBX3 over-expressing 21NT cells for extravasation. These TBX3-induced changes were associated with increased vimentin expression, but minimal change in e-cadherin expression. Overall, these results indicate that TBX3 (iso1 or iso2) expression can promote progression in a model of early breast cancer, by altering cell properties involved in cell survival/colony formation and invasiveness. Citation Format: Connor D. MacMillan, Hon S. Leong, Allen G. Clifford, Milica Krstic, Siddika Pardhan, David W. Dales, Carl O. Postenka, Ann F. Chambers, Alan B. Tuck. The transcriptional regulator TBX3 promotes progression of cells representing early premalignant breast cancer. [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 4055. doi:10.1158/1538-7445.AM2014-4055

Breast cancer metastasis suppressor 1 (BRMS1) inhibits the ability of multiple human and murine cancer cell lines to metastasize to lymph nodes, bones and lungs. Comparison of mRNA expression in metastatic MDA‐MB‐435 human carcinoma cells (435) and metastasis‐suppressed BRMS1 transfectants (435/BRMS1) showed a marked (>90%) reduction of osteopontin (OPN) mRNA and protein expression in BRMS1‐overexpressing cells. OPN expression is associated with disease progression in patients, with higher levels of OPN produced by cancer cells associated with poorer patient survival. Furthermore, OPN has been suggested to promote survival of cancer cells in response to stress, although the mechanisms by which this may occur remain poorly understood. This study tested the hypothesis that re‐expression of OPN in metastasis‐suppressed 435/BRMS1 cells would reverse metastasis suppression and confer protection from stress‐induced apoptosis. A stable pooled population of OPN overexpressing 435/BRMS1 cells was created (435/BRMS1/OPN). OPN re‐expression did not affect in vitro cell growth rates; however, increased anchorage independent growth/survival and protection from hypoxia‐induced apoptosis was observed ( p < 0.05). In vivo , OPN re‐expression in BRMS1 transfected cells did not affect in vivo primary tumor growth but did increase the incidence of spontaneous metastasis to lymph nodes and lungs in mice. These novel findings suggest that OPN downregulation by BRMS1 may be responsible, at least in part, for BRMS1‐mediated metastasis suppression by sensitizing cancer cells to stress induced apoptosis. These studies clarify one mechanism by which BRMS1 can suppress metastasis. © 2008 Wiley‐Liss, Inc.

The majority of breast cancer related deaths occur as a result of metastasis. The failure of effective treatments for metastasis is the underlying cause for this. Much remains unknown about the process of metastasis and how best to prevent or treat metastatic breast cancer. Therefore, a better understanding of the metastatic process is needed in order to determine effective therapeutic interventions to either eradicate, or slow down metastatic outgrowth of breast cancer. Metastasis is an inefficient process, however the ability of only a small number of cells to complete this process may have serious, life-threatening consequences. Little is known about whether expression of the metastasis suppressor breast cancer metastasis suppressor 1 (BRMS1) can suppress metastatic outgrowth in different organs in multiple experimental models of metastasis, or what effect BRMS1 expression has on the various steps in metastatic cascade. In this study we investigated the effect of BRMS1 expression on organ-specific metastasis. In addition, the steps in metastasis that are inhibited by BRMS1-expression were determined. In vivo, BRMS1 expression reduced metastatic burden to liver, bone, brain, and lung in mice by at least 75% (P<0.05). Detailed quantitative analysis of the metastatic process in lung showed that BRMS1 expression significantly reduced the numbers of solitary single cells that survive after initial arrest within the lung microvasculature, and also inhibited the initiation of growth subsequent to arrest. In vitro, BRMS1 expression decreased cancer cell survival under stress conditions (hypoxia), increased anoikis, and decreased the ability of cancer cells to adhere. These novel findings demonstrate that BRMS1 is a potent suppressor of metastasis in multiple organs, and identify two steps in the metastatic process that are sensitive to inhibition by BRMS1.

Survival following diagnosis of liver metastasis remains poor and improved treatment strategies to combat liver metastases are needed. Synthetic triterpenoids, including 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Imidazolide or CDDO-Im), have been shown to inhibit primary tumor growth and lung metastasis in experimental models. Oral administration of CDDO-Im results in relatively high liver concentrations, suggesting that CDDO-Im may provide an approach to treatment of liver metastases. Here we assessed the effect of CDDO-Im on liver metastasis, using B16F1 (mouse melanoma) and HT-29 (human colon carcinoma) cells. In vitro, nanomolar concentrations of CDDO-Im arrested proliferation or induced cell death in both cell lines. In vivo, cells were injected via a surgically exposed mesenteric vein to target cells to the liver of mice. Mice were then treated with CDDO-Im (800 mg/kg diet) or vehicle control. Livers were removed at endpoint and metastatic burden was quantified by standard histology. In addition, a novel whole liver magnetic resonance imaging (MRI) technique was used to assess the effect of CDDO-Im on growing metastases as well as on non-dividing, solitary cancer cells present in the same livers. CDDO-Im treatment significantly decreased liver metastasis burden in both HT-29 (n = 8 treated, 10 control) and B16F1 (n = 15 treated, 16 control) injected mice (>60%, P < 0.05), but did not reduce the numbers of solitary B16F1 cancer cells (hypo-intensity) in the same livers (P = 0.9). This study demonstrates that CDDO-Im may be useful for the treatment metastatic liver disease as it successfully inhibits growth of actively proliferating liver metastases.

Proc Amer Assoc Cancer Res, Volume 47, 2006 3974 Introduction: Metastasis is the main cause of death in breast cancer. A better understanding of the metastatic process is needed if these deaths are to be prevented. Breast Cancer Metastasis Suppressor 1 gene (BRMS1) inhibits the ability of multiple human and murine cancer cell lines to metastasize to lymph node (LN) and lung. Comparison of mRNA expression in metastatic MDA-MB-435 human breast carcinoma cells (435) and metastasis-suppressed BRMS1 transfectants (435/BRMS1) showed a marked (>90%) reduction of osteopontin (OPN) mRNA and protein expression. OPN expression is associated with disease progression in patients, with higher levels of OPN produced by cancer cells associated with poorer patient survival. This work tests the hypothesis that restoration of OPN levels in 435/BRMS1 would be accompanied by regaining of metastatic capability. Methods: Neomycin-resistant 435/BRMS1 cells were transfected with either full-length OPN cDNA or a vector only plasmid (pcDNA3.1+) and single colonies were selected by hygromycin resistance. Stable colonies were selected to create pooled populations of OPN over-expressing (435/BRMS1/OPN) and vector control (435/BRMS1/VC) cell lines, which were assessed in vitro (colony formation in soft agar). In vivo , parental 435, 435/BRMS1/VC, and 435/BRMS1/OPN cells were implanted into the left thoracic mammary fat pad of female athymic mice. Primary tumors were allowed to grow for 8 wk prior to resection. The mice were then left for a further 8 weeks or until moribund. Metastatic tumor burden (tumor area/organ total area) was calculated from analysis of histological slides using Image software from the National Institutes of Health. Results: Re-expression of OPN in BRMS1 expressing breast cancer cells was achieved and resulted in increased number and size of colonies in soft agar. Quantification of total metastatic burden within LN and lung revealed that re-expression of OPN significantly increased (p < 0.05) metastatic tumor burden within the LN but not in lung. In LN, metastatic burden was 76% for 435, 2% for 435/BRMS1, and 35% for 435/BRMS1/OPN cells. In lung, metastatic burden was 38% for 435, 1% for 435/BRMS1, and 3% for 435/BRMS1/OPN cells. Conclusion: Re-expression of OPN in 435/BRMS1 cells increased number and size of colonies in soft agar, thus OPN down-regulation by BRMS1 may be responsible for decreased anchorage independent growth in vitro . Mice injected with 435/BRMS1/OPN cells developed significantly more LN, but not lung, metastases than 435/BRMS1 cells. Thus, OPN down-regulation by BRMS1 may be responsible, in part, for decreased LN metastatic burden but did not appear to be involved in decreased lung metastatic burden. These novel studies clarify one mechanism by which BRMS1 can suppress metastasis, which may lead to new therapeutic approaches for suppressing breast cancer metastasis based on this understanding.