Targeting the Vav1/miR­29b axis as a potential approach for treating selected molecular subtypes of triple­negative breast cancer.

MicroRNA (miR)­29b has been reported to play a controversial role in breast cancer, particularly triple­negative breast cancer (TNBC). Based on our previous data revealing that the PU.1­mediated expression of miR­29b in cells from acute myeloid leukemia is sustained by Vav1, the potential role of this multidomain protein in modulating miR­29b levels in breast tumor cells, in which Vav1 is ecstopically expressed and shows a nuclear accumulation, was investigated. Breast cancer cell lines with various phenotypes and patient­derived xenograft­derived TNBC cells were subjected to Vav1 modulation and reverse transcription quantitative PCR of miR­29b levels. The recruitment of CCAAT enhancer binding protein α (CEBPα) to miR­29b promoters was investigated by quantitative chromatin immunoprecipitation assays. It was found that Vav1 was essential for the recovery of mature miR­29b in breast cancer cell lines, and that it promoted the expression of the miRNA in TNBC cells of the mesenchymal molecular subtype by sustaining the transcription of the miR­29b1/a cluster mediated by CEBPα. The present results suggest that Vav1 is a crucial modulator of miR­29b expression in breast tumor cells, and this finding may help identify strategies that may be useful in the management of TNBC by targeting the Vav1/miR­29b axis, as there is a lack of molecular­based treatments for TNBC.

Ectopic expression of PLC-ß2 in non-invasive breast tumor cells plays a protective role against malignant progression and is correlated with the deregulation of miR-146a.

Cells in non-invasive breast lesions are widely believed to possess molecular alterations that render them either susceptible or refractory to the acquisition of invasive capability. One such alteration could be the ectopic expression of the ß2 isoform of phosphoinositide-dependent phospholipase C (PLC-ß2), known to counteract the effects of hypoxia in low-invasive breast tumor-derived cells. Here, we studied the correlation between PLC-ß2 levels and the propensity of non-invasive breast tumor cells to acquire malignant features. Using archival FFPE samples and DCIS-derived cells, we demonstrate that PLC-ß2 is up-regulated in DCIS and that its forced down-modulation induces an epithelial-to-mesenchymal shift, expression of the cancer stem cell marker CD133, and the acquisition of invasive properties. The ectopic expression of PLC-ß2 in non-transformed and DCIS-derived cells is, to some extent, dependent on the de-regulation of miR-146a, a tumor suppressor miRNA in invasive breast cancer. Interestingly, an inverse relationship between the two molecules, indicative of a role of miR-146a in targeting PLC-ß2, was not detected in primary DCIS from patients who developed a second invasive breast neoplasia. This suggests that alterations of the PLC-ß2/miR-146a relationship in DCIS may constitute a molecular risk factor for the appearance of new breast lesions. Since neither traditional classification systems nor molecular characterizations are able to predict the malignant potential of DCIS, as is possible for invasive ductal carcinoma (IDC), we propose that the assessment of the PLC-ß2/miR-146a levels at diagnosis could be beneficial for identifying whether DCIS patients may have either a low or high propensity for invasive recurrence.

Protective role of all-trans retinoic acid (ATRA) against hypoxia-induced malignant potential of non-invasive breast tumor derived cells.

BACKGROUND: The presence of hypoxic areas is common in all breast lesions but no data clearly correlate low oxygenation with the acquisition of malignant features by non-invasive cells, particularly by cells from ductal carcinoma in situ (DCIS), the most frequently diagnosed tumor in women. METHODS: By using a DCIS-derived cell line, we evaluated the effects of low oxygen availability on malignant features of non-invasive breast tumor cells and the possible role of all-trans retinoic acid (ATRA), a well-known anti-leukemic drug, in counteracting the effects of hypoxia. The involvement of the ß2 isoform of PI-PLC (PLC-ß2), an ATRA target in myeloid leukemia cells, was also investigated by specific modulation of the protein expression. RESULTS: We demonstrated that moderate hypoxia is sufficient to induce, in DCIS-derived cells, motility, epithelial-to-mesenchymal transition (EMT) and expression of the stem cell marker CD133, indicative of their increased malignant potential. Administration of ATRA supports the epithelial-like phenotype of DCIS-derived cells cultured under hypoxia and keeps down the number of CD133 positive cells, abrogating almost completely the effects of poor oxygenation. We also found that the mechanisms triggered by ATRA in non-invasive breast tumor cells cultured under hypoxia is in part mediated by PLC-ß2, responsible to counteract the effects of low oxygen availability on CD133 levels. CONCLUSIONS: Overall, we assigned to hypoxia a role in increasing the malignant potential of DCIS-derived cells and we identified in ATRA, currently used in treatment of acute promyelocytic leukemia (APL), an agonist potentially useful in preventing malignant progression of non-invasive breast lesions showing hypoxic areas.

Vav1 is necessary for PU.1 mediated upmodulation of miR-29b in acute myeloid leukaemia-derived cells.

It has been recently demonstrated that high pre-treatment levels of miR-29b positively correlated with the response of patients with acute myeloid leukaemia (AML) to hypomethylating agents. Upmodulation of miR-29b by restoring its transcriptional machinery appears indeed a tool to improve therapeutic response in AML. In cells from acute promyelocytic leukaemia (APL), miR-29b is regulated by PU.1, in turn upmodulated by agonists currently used to treat APL. We explored here the ability of PU.1 to also regulate miR-29b in non-APL cells, in order to identify agonists that, upmodulating PU.1 may be beneficial in hypomethylating agents-based therapies. We found that PU.1 may regulate miR-29b in the non-APL Kasumi-1 cells, showing the t(8;21) chromosomal rearrangement, which is prevalent in AML and correlated with a relatively low survival. We demonstrated that the PU.1-mediated contribution of the 2 miR-29b precursors is cell-related and almost completely dependent on adequate levels of Vav1. Nuclear PU.1/Vav1 association accompanies the transcription of miR-29b but, at variance with the APL-derived NB4 cells, in which the protein is required for the association of PU.1 with both miRNA promoters, Vav1 is part of molecular complexes to the PU.1 consensus site in Kasumi-1. Our results add new information on the transcriptional machinery that regulates miR-29b expression in AML-derived cells and may help in identifying drugs useful in upmodulation of this miRNA in pre-treatment of patients with non-APL leukaemia who can take advantage from hypomethylating agent-based therapies.

Up-modulation of PLC-ß2 reduces the number and malignancy of triple-negative breast tumor cells with a CD133+/EpCAM+ phenotype: a promising target for preventing progression of TNBC.

BACKGROUND: The malignant potential of triple negative breast cancer (TNBC) is also dependent on a sub-population of cells with a stem-like phenotype. Among the cancer stem cell markers, CD133 and EpCAM strongly correlate with breast tumor aggressiveness, suggesting that simultaneous targeting of the two surface antigens may be beneficial in treatment of TNBC. Since in TNBC-derived cells we demonstrated that PLC-ß2 induces the conversion of CD133high to CD133low cells, here we explored its possible role in down-modulating the expression of both CD133 and EpCAM and, ultimately, in reducing the number of TNBC cells with a stem-like phenotype. METHODS: A magnetic step-by-step cell isolation with antibodies directed against CD133 and/or EpCAM was performed on the TNBC-derived MDA-MB-231 cell line. In the same cell model, PLC-ß2 was over-expressed or down-modulated and cell proliferation and invasion capability were evaluated by Real-time cell assays. The surface expression of CD133, EpCAM and CD44 in the different experimental conditions were measured by multi-color flow cytometry immunophenotyping. RESULTS: A CD133+/EpCAM+ sub-population with high proliferation rate and invasion capability is present in the MDA-MB-231 cell line. Over-expression of PLC-ß2 in CD133+/EpCAM+ cells reduced the surface expression of both CD133 and EpCAM, as well as proliferation and invasion capability of this cellular subset. On the other hand, the up-modulation of PLC-ß2 in the whole MDA-MB-231 cell population reduced the number of cells with a CD44+/CD133+/EpCAM+ stem-like phenotype. CONCLUSIONS: Since selective targeting of the cells with the highest aggressive potential may have a great clinical importance for TNBC, the up-modulation of PLC-ß2, reducing the number of cells with a stem-like phenotype, may be a promising goal for novel therapies aimed to prevent the progression of aggressive breast tumors.

Correlation between Slug transcription factor and miR-221 in MDA-MB-231 breast cancer cells.

BACKGROUND: Breast cancer and its metastatic progression is mainly directed by epithelial to mesenchymal transition (EMT), a phenomenon supported by specific transcription factors and miRNAs. METHODS: In order to investigate a possible correlation between Slug transcription factor and miR-221, we performed Slug gene silencing in MDA-MB-231 breast cancer cells and evaluated the expression of genes involved in supporting the breast cancer phenotype, using qRT-PCR and Western blot analysis. Chromatin immunoprecipitation and wound healing assays were employed to determine a functional link between these two molecules. RESULTS: We showed that Slug silencing significantly decreased the level of miR-221 and vimentin, reactivated Estrogen Receptor α and increased E-cadherin and TRPS1 expression. We demonstrated that miR-221 is a Slug target gene, and identified a specific region of miR-221 promoter that is transcriptionally active and binds the transcription factor Slug "in vivo". In addition, we showed that in Slug-silenced cells, wich retained residual miR-221 (about 38%), cell migration was strongly inhibited. Cell migration was inhibited, but to a less degree, following complete knockdown of miR-221 expression by transfection with antagomiR-221. CONCLUSIONS: We report for the first time evidence of a correlation between Slug transcription factor and miR-221 in breast cancer cells. These studies suggest that miR-221 expression is, in part, dependent on Slug in breast cancer cells, and that Slug plays a more important role than miR-221 in cell migration and invasion.