Prolactin (PRL) hormone plays an important role in the development of the mammary gland and terminal differentiation of the mammary epithelial cells. While initial studies suggested that PRL may contribute to the development of breast cancer through PRL/prolactin receptor (PRLR) autocrine function, mounting evidence indicate a different role for PRL, highlighting this hormone as a regulator of epithelial plasticity and as a potential tumor suppressor. To gain further insights into the role of PRL in human breast carcinogenesis, immunohistochemistry analyses of PRLR protein expression levels using tissue microarray of 102 cases were done in comparison with various clinical/pathologic parameters and molecular subtypes. In addition, gene expression level of PRLR was also evaluated in relation to intrinsic molecular subtypes, tumor grade, and patient outcome using GOBO database for 1881 breast cancer patients. Interestingly, PRLR expression was found to be significantly downregulated in invasive breast cancer (21.4%) in comparison with normal/benign (80%) and in situ carcinoma (60%) (P=0.003498). Moreover, PRLR expression was associated with lymph node negativity and low-grade well-differentiated tumors. PRLR expression was strongest in luminal A subtype, and was virtually undetectable in the worse prognosis triple-negative breast cancer subtype (P=0.00001). Furthermore, PRLR expression was independent of ER, PR, HER-2, and P53 status. Finally, PRLR expression was significantly (P<0.01) associated with prolonged distant metastasis–free survival in breast cancer patients. In conclusion, our results highlight PRLR as an independent predictor of favorable prognosis in human breast cancer.

Triple negative breast cancer (TNBC) is a heterogeneous disease associated with poor clinical outcome and lack of targeted therapy. Here we show that prolactin (PRL) and its signaling pathway serve as a sub-classifier and predictor of pro-differentiation therapy in TNBC. Using immunohistochemistry and various gene expression in silica analyses we observed that prolactin receptor (PRLR) protein and mRNA levels are down regulated in TNBC cases. In addition, examining correlation of PRLR gene expression with metagenes of TNBC subtypes (580 cases), we found that PRLR gene expression sub-classifies TNBC patients into a new subgroup (TNBC-PRLR) characterized by epithelial-luminal differentiation. Importantly, gene expression of PRL signaling pathway components individually (PRL, PRLR, Jak2 and Stat5a), or as a gene signature is able to predict TNBC patients with significantly better survival outcomes. As PRL hormone is a druggable target we determined the biological role of PRL in TNBC biology. Significantly, restoration/activation of PRL pathway in TNBC cells representative of mesenchymal or TNBC-PRLR subgroups led to induction of epithelial phenotype and suppression of tumorigenesis. Altogether, these results offer potential new modalities for TNBC stratification and development of personalized therapy based on PRL pathway activation.

Triple-negative breast cancer (TNBC) accounts for ~20% of all breast cancer cases. The management of TNBC represents a challenge due to its aggressive phenotype, heterogeneity and lack of targeted therapy. Loss of cell differentiation and enrichment with breast cancer stem-like cells (BCSC) are features of TNBC contributing to its aggressive nature. Here, we found that treatment of TNBC cells with PRL significantly depletes the highly tumorigenic BCSC subpopulations CD44⁺/CD24⁻and ALDH⁺and differentiates them to the least tumorigenic CD44⁻/CD24⁻and ALDH⁻phenotype with limited tumorsphere formation and self-renewal capacities. Importantly, we found PRL to induce a heterochromatin phenotype marked by histone H3 lysine 9 trimethylation (H3K9me3) and accompanied by ultra-structural cellular architecture associated with differentiation and senescence rendering the cells refractory to growth signals. Crucially, we found PRL to mediate these effectsin vivoin a pre-clinical animal xenograft of TNBC controlling tumor growth. These results reveal that the lactogenic hormone PRL may exert its anti-tumorigenic effects on TNBC through cellular reprogramming indicative of differentiation resulting in the depletion of BCSCs and restricting tumorigenesis.

Breast cancers characterized by HER2 overexpression, belong to HER-2 enriched or luminal B subtypes, are frequently associated with higher incidence of tumor recurrence and therapeutic failure. These aggressive features have been attributed to the presence of cancer stem-like cell subpopulations known to have high tumor initiation, self -renewal capacities and high metastatic potential. Depleting these stem-like cells in these tumors therefore might help in improving therapeutic response and patient outcome. Here we used human breast cancer cells representative of HER2- enriched and luminal B subtypes as well as purified ALDH-positive stem-like cell subpopulation for in vitro cell viability, proliferation, tumorshpere formation analyses and gene expression studies. In addition, we used a pre-clinical xenograft HER2 mouse model (NOD/SCID mice) for in vivo tumorigenesis assessment. Furthermore, patient survival outcomes were evaluated using in silico bioinformatics analyses of publicly available datasets. Our results indicate that prolactin (PRL) exerts anti-tumorigenic effects in HER-2 positive breast cancer cells. Importantly, PRL caused a significant reduction in ALDHhi stem-like subpopulation, as well as their viability and tumorsphere formation capacity. Molecularly we found PRL to suppress gene expression of markers involved in stemness, tumor initiation, drug resistance and poor patient outcome found to be enriched in the ALDHhi stem-like subpopulation. Furthermore, we show PRL to impede tumor growth of HER-2 xenografts and to suppress expression of Ki67 proliferative marker. Finally, we found PRL pathway gene signature to correlate with favorable patient outcomes in HER-2 and luminal B breast cancer patients. Together these results emphasize an anti-tumorigenic role with a potential therapeutic value for PRL in HER-2 and luminal B breast cancer subtypes targeting the cancer stem-like cells.

Poly (ADP-ribose) polymerase inhibitors (PARPi) have become the first targeted therapies available in the treatment of patients with high-grade serous ovarian cancer (HGSOC). We recently described a significant reduction in PARP1 protein levels in vitro and in vivo in patients treated with standard carboplatinum-paclitaxel chemotherapy, raising the question whether the sequence of treatment used today with chemotherapy followed by PARPi is optimal. In this study, we aim to evaluate if the sequence of PARPi followed by chemotherapy could be more beneficial. BRCA1-mutated (UWB1.287, SNU-251), epigenetically-silenced (OVCAR8), and wild-type (SKOV3, A2780PAR & A2780CR) ovarian cancer cell lines were exposed to clinically relevant doses of PARPi followed by different doses of standard chemotherapy and compared to the inverse treatment. The therapeutic efficacy was assessed using colony formation assays. Flow cytometry was used to evaluate cell apoptosis rate and the changes in cell cycle. Finally, apoptotic and cell cycle protein expression was immunodetected using western blot. Exposure to PARPi prior to standard chemotherapy sensitized BRCA1-mutated or epigenetically-silenced BRCA1 cell lines to lower doses of chemotherapy. Similar results were observed in BRCA1 wild-type and cell lines in which BRCA1 functionality was restored. Moreover, this treatment increased the apoptotic rate in these cell lines. Pre-treatment with PARPi followed by standard chemotherapy in vitro is more efficient in growth inhibition and induction of apoptosis compared to the administration of standard chemotherapy followed by PARPi.