Ectopic expression of PLC‐β2 in non‐invasive breast tumor cells plays a protective role against malignant progression and is correlated with the deregulation of miR‐146a

Abstract

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 β2 isoform of phosphoinositide‐dependent phospholipase C (PLC‐β2), known to counteract the effects of hypoxia in low‐invasive breast tumor‐derived cells. Here, we studied the correlation between PLC‐β2 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‐β2 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‐β2 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‐β2, was not detected in primary DCIS from patients who developed a second invasive breast neoplasia. This suggests that alterations of the PLC‐β2/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‐β2/miR‐146a levels at diagnosis could be beneficial for identifying whether DCIS patients may have either a low or high propensity for invasive recurrence.

Vav1 Selectively Down-Regulates Akt2 through miR-29b in Certain Breast Tumors with Triple Negative Phenotype

“…For specific modulation of miR-29b-3p, transient transfections were performed with a synthetic inhibitor or mimic (miRVana miRNA, Thermo Fisher Scientific, Waltham, MA, USA), as previously reported [ 25 ].…”

“…Sections of breast tumor xenografts were laser micro-dissected (LMD) by using the LEICA LMD 6500 laser microdissector (Leica Microsystems, Wetzlar, Germany), as previously reported [ 25 ]. For each sample, areas of 2–20 × 10 6 μm 2 were selected and micro-dissected samples were subjected to microRNAs extraction by using the miRNeasy FFPE kit (Qiagen, Milan, Italy).…”

Targeting the Vav1/miR‑29b axis as a potential approach for treating selected molecular subtypes of triple‑negative breast cancer

“…The BT-474 cell line was cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS), 1 mM Na pyruvate, and 0.01 mg/ml bovine insulin. MCF7, MDA-MB-453, MDA-MB-468 and MDA-MB-231 cell lines were maintained in DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (17)(18)(19).…”

“…miR-29b and CEBPα expression were evaluated by RT-qPCR using TaqMan Assays (ID 000413; ID Hs05650633_s1: Thermo Fisher Scientific, Inc), as previously described (16,19,20). miR-29b and CEBPα expression levels were normalized to U6 snRNA (ID 001973, Thermo Fisher Scientific, Inc.) and to RPL13A (ID Hs03043885_g1, Thermo Fisher Scientific, Inc.), respectively.…”

CD133 in Breast Cancer Cells: More than a Stem Cell Marker

“…PLC- β 2 is ectopically expressed and regulates the number of cells expressing CD133 also in the noninvasive MCF10DCIS cells [75]. In the same cell model, the administration of all trans retinoic acid (ATRA), currently used in the management of acute promyelocytic leukemia [76] in which it induces the expression of PLC- β 2 [77], counteracts the effects of hypoxia on CD133 expression by up-modulating the PLC isozyme [64].…”