Pancreatic progenitors (PPs) co-expressing the two transcription factors (TFs) PDX1 and NKX6.1 are recognized as the indispensable precursors of functional pancreatic β cells. Here, we aimed to establish an efficient protocol for maximizing generation of PDX1+/NKX6.1+ PPs from human pluripotent stem cells (hPSCs). In order to enhance the PDX1+/NKX6.1+ population, we manipulated in vitro culture conditions during differentiation by dissociating densely formed endodermal cells and re-plating them at different densities. These dissociated cells were subjected to an augmented duration of retinoid and fibroblast growth factor (FGF)10 signaling to induce higher PDX1 and NKX6.1 expression. Our optimized protocol dramatically increased the expression of NKX6.1, leading to an increase in the proportion of PDX1+/NKX6.1+ progenitors (~90%) in monolayer, higher than the previously published protocols, as well as upregulated key TFs controlling pancreatic development. The improved efficiency of pancreatic differentiation was complemented by an inhibited hepatic specification and an increased proliferation of NKX6.1+ cells. Interestingly, we were able to enrich a novel PDX1–/NKX6.1+ population by manipulating the re-plating density; these oriented themselves in three-dimensional clusters. Further differentiation validated the ability of our PDX1+/NKX6.1+ progenitors to generate NGN3+ endocrine progenitors. We provide a novel technique that facilitates appropriate cellular rearrangement in monolayer culture to yield a high proportion of PDX1+/NKX6.1+ PPs with an elevated self-replicating capacity, thereby aiding scalable production of functional β cells from hPSCs in vitro. Our innovative method also enriches a novel NKX6.1+/PDX1– population, with characteristics of proposed endocrine precursors, allowing further studies on deciphering routes to β-cell development.

We examined the potential value of the natural killer (NK) cell line; NK-92, as immunotherapy tool for breast cancer (BC) treatment and searched for biomarker(s) of sensitivity to NK-92-mediated cytotoxicity. The cytotoxic activity of NK-92 cells towards one breast precancerous and nine BC cell lines was analyzed using calcein-AM and degranulation assays. The molecules associated with NK-92-responsiveness were determined by differential gene expression analysis using RNA-sequencing and validated by RT-PCR, immunostaining and flow cytometry. NK-target interactions and immunological synapse formation were assessed by fluorescence microscopy. Potential biomarker expression was determined by IHC in 99 patient-derived BC tissues and 10 normal mammary epithelial tissues. Most (8/9) BC cell lines were resistant while only one BC and the precancerous cell lines were effectively killed by NK-92 lymphocytes. NK-92-sensitive target cells specifically expressed CD56, which ectopic expression in CD56-negative BC cells induced their sensitivity to NK-92-mediated killing, suggesting that CD56 is not only a biomarker of responsiveness but actively regulates NK function. CD56 adhesion molecules which are also expressed on NK cells accumulate at the immunological synapse enhancing NK-target interactions, cytotoxic granzyme B transfer from NK-92 to CD56-expressing target cells and induction of caspase 3 activation in targets. Interestingly, CD56 expression was found to be reduced in breast tumor tissues (36%) with strong inter- and intratumoral heterogeneity in comparison to normal breast tissues (80%). CD56 is a potential predictive biomarker for BC responsiveness to NK-92-cell based immunotherapy and loss of CD56 expression might be a mechanism of escape from NK-immunity.

About 70% of human breast cancers express and are dependent for growth on estrogen receptor α (ERα), and therefore are sensitive to antiestrogen therapies. However, progression to an advanced, more aggressive phenotype is associated with acquisition of resistance to antiestrogens and/or invasive potential. In this study, we highlight the role of the serine/threonine‐protein kinase D1 (PKD1) in ERα‐positive breast cancers. Growth of ERα‐positive MCF‐7 and MDA‐MB‐415 human breast cancer cells was assayed in adherent or anchorage‐independent conditions in cells overexpressing or depleted for PKD1. PKD1 induces cell growth through both an ERα‐dependent manner, by increasing ERα expression and cell sensitivity to 17β‐estradiol, and an ERα‐independent manner, by reducing cell dependence to estrogens and conferring partial resistance to antiestrogen ICI 182,780. PKD1 knockdown in MDA‐MB‐415 cells strongly reduced estrogen‐dependent and independent invasion. Quantification of PKD1 mRNA levels in 38 cancerous and non‐cancerous breast cell lines and in 152 ERα‐positive breast tumours from patients treated with adjuvant tamoxifen showed an association between PKD1 and ERα expression in 76.3% (29/38) of the breast cell lines tested and a strong correlation between PKD1 expression and invasiveness (P < 0.0001). In tamoxifen‐treated patients, tumours with high PKD1 mRNA levels (n = 77, 50.66%) were significantly associated with less metastasis‐free survival than tumours with low PKD1 mRNA expression (n = 75, 49.34%; P = 0.031). Moreover, PKD1 mRNA levels are strongly positively associated with EGFR and vimentin levels (P < 0.0000001). Thus, our study defines PKD1 as a novel attractive prognostic factor and a potential therapeutic target in breast cancer.

Melanoma is a highly metastatic type of cancer that is resistant to all standard anticancer therapies and thus has a poor prognosis. Therefore, metastatic melanoma represents a significant clinical problem and requires novel and effective targeted therapies. The protein kinase C (PKC) family comprises multiple isoforms of serine/threonine kinases that possess distinct roles in cancer development and progression. In this study, we determined whether inhibition of PKC could revert a major process required for melanoma progression and metastasis; i.e. the E- to N-cadherin switch. The cadherin switch was analyzed in different patient-derived primary tumors and their respective metastatic melanoma cells to determine the appropriate cellular model (aggressive E-cadherin-negative/N-cadherin-positive metastasis-derived melanoma cells). Next, PKC inhibition in two selected metastatic melanoma cell lines, was performed by using either pharmacological inhibitors (Gö6976 and Gö6983) or stable lentiviral shRNA transduction. The expression of E-cadherin and N-cadherin was determined by western blot. The consequences of cadherin switch reversion were analyzed: cell morphology, intercellular interactions, and β-catenin subcellular localization were analyzed by immunofluorescence labeling and confocal microscopy; cyclin D1 expression was analyzed by western blot; cell metastatic potential was determined by anchorage-independent growth assay using methylcellulose as semi-solid medium and cell migration potential by wound healing and transwell assays. Gö6976 but not Gö6983 reversed the E- to N-cadherin switch and as a consequence induced intercellular interactions, profound morphological changes from elongated mesenchymal-like to cuboidal epithelial-like shape, β-catenin translocation from the nucleus to the plasma membrane inhibiting its oncogenic function, and reverting the metastatic potential of the aggressive melanoma cells. Comparison of the target spectrum of these inhibitors indicated that these observations were not the consequence of the inhibition of conventional PKCs (cPKCs), but allowed the identification of a novel serine/threonine kinase, i.e. protein kinase Cμ, also known as protein kinase D1 (PKD1), whose specific inhibition allows the reversion of the metastatic phenotype in aggressive melanoma. In conclusion, our study suggests, for the first time, that while cPKCs don’t embody a pertinent therapeutic target, inhibition of PKD1 represents a novel attractive approach for the treatment of metastatic melanoma.

Human pluripotent stem cells (hPSCs) can provide unlimited supply for mesenchymal stem cells (MSCs) and adipocytes that can be used for therapeutic applications. Here we developed a simple and highly efficient all-trans-retinoic acid (RA)-based method for generating an off-the-shelf and scalable number of human pluripotent stem cell (hPSC)-derived MSCs with enhanced adipogenic potential. We showed that short exposure of multiple hPSC lines (hESCs/hiPSCs) to 10 μM RA dramatically enhances embryoid body (EB) formation through regulation of genes activating signaling pathways associated with cell proliferation, survival and adhesion, among others. Disruption of cell adhesion induced the subsequent differentiation of the highly expanded RA-derived EB-forming cells into a pure population of multipotent MSCs (up to 1542-fold increase in comparison to RA-untreated counterparts). Interestingly, the RA-derived MSCs displayed enhanced differentiation potential into adipocytes. Thus, these findings present a novel RA-based approach for providing an unlimited source of MSCs and adipocytes that can be used for regenerative medicine, drug screening and disease modeling applications.