Chemoresistance to cisplatin is a principal cause of treatment failure and mortality of advanced bladder cancer (BC). The underlying mechanisms remain unclear, which hinders the development of preventive strategies. Recent data indicate that pyruvate kinase M2 (PKM2), a glycolytic enzyme for Warburg effect, is strongly upregulated in BC. This study explores the role of PKM2 in chemoresistance and whether inhibiting PKM2 augments the chemosensitivity to cisplatin and reduces BC growth and progression. We found that Shikonin binds PKM2 and inhibits BC cell survival in a dose-dependent but pyruvate kinase activity-independent manner. Down-regulation of PKM2 by shRNA blunts cellular responses to shikonin but enhances the responses to cisplatin. Shikonin and cisplatin together exhibit significantly greater inhibition of proliferation and apoptosis than when used alone. Induced cisplatin-resistance is strongly associated with PKM2 overexpression, and cisplatin-resistant cells respond sensitively to shikonin. In syngeneic mice, shikonin and cisplatin together, but not as single-agents, markedly reduces BC growth and metastasis. Based on these data, we conclude that PKM2 overexpression is a key mechanism of chemoresistance of advanced BC to cisplatin. Inhibition of PKM2 via RNAi or chemical inhibitors may be a highly effective approach to overcome chemoresistance and improve the outcome of advanced BC.

The conversion of precancerous lesions to full-fledged cancers requires the affected cells to surpass certain rate-limiting steps. We recently showed that activation of HRAS proto-oncogene in urothelial cells of transgenic mice causes simple urothelial hyperplasia (SUH) which is persistent and whose transition to low-grade papillary urothelial carcinoma (UC) must undergo nodular urothelial hyperplasia (NUH). We hypothesized that NUH, which has acquired fibrovascular cores, plays critical roles in mesenchymal-to-epithelial signaling, breaching the barriers of urothelial tumor initiation. Using proteomics involving two-dimensional gel electrophoresis, immunoblotting with pan-phosphotyrosine antibody and MALDI-mass spectrometry, we identified isoform 2 of pyruvate kinase (PKM2) as the major tyrosine-phosphorylated protein switched on during NUH. We extended this finding using specimens from transgenic mice, human UC and UC cell lines, establishing that PKM2, but not its spliced variant PKM1, was over-expressed in low-grade and, more prominently, high-grade UC. In muscle-invasive UC, PKM2 was co-localized with cytokeratins 5 and 14, UC progenitor markers. Specific inhibition of PKM2 by siRNA or shRNA suppressed UC cell proliferation via increased apoptosis, autophagy and unfolded protein response. These results strongly suggest that PKM2 plays an important role in the genesis of low-grade non-invasive and high-grade invasive urothelial carcinomas.

The purpose of this investigation was to determine the role of extracellular vesicles (EVs), released from articular chondrocytes in a physiological or pathological state, in cell–cell communication with other articular chondrocytes or chondrocyte precursor cells. The conditioned medium from interleukin‐1β (IL‐1β)‐treated human articular chondrocytes stimulated catabolic events and inhibited type II collagen expression in articular chondrocytes to a much greater degree than medium from IL‐1β‐treated chondrocytes after complete removal of EVs. The vehicle‐treated and IL‐1β‐treated human articular chondrocytes released EVs of similar size; however, the number of EVs released by IL‐1β‐treated chondrocytes was markedly higher than the number of EVs released from the vehicle‐treated cells. Furthermore, our findings demonstrate that similar to medium from IL‐1β‐treated chondrocytes containing EVs, EVs isolated from medium of IL‐1β‐treated chondrocytes stimulated catabolic events in articular chondrocytes, whereas EVs isolated from the medium of vehicle‐treated chondrocytes inhibited catabolic events and increased messenger RNA levels of aggrecan and type II collagen in IL‐1β‐treated chondrocytes. Furthermore, the medium containing EVs from vehicle‐treated articular chondrocytes or EVs isolated from this medium stimulated chondrogenesis of C3H10T1/2 cells, whereas medium containing EVs from IL‐1β‐treated chondrocytes or EVs isolated from this medium inhibited chondrogenesis. Our findings suggest that EVs released by articular chondrocytes play a key role in the communication between joint cells and ultimately in joint homeostasis, maintenance, pathology, and repair. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:731‐739, 2020

Mesenchymal stem cells (MSCs) obtained from various sources, including bone marrow, have been proposed as a therapeutic strategy for the improvement of tissue repair/regeneration, including the repair of cartilage defects or lesions. Often the highly inflammatory environment after injury or during diseases, however, greatly diminishes the therapeutic and reparative effectiveness of MSCs. Therefore, the identification of novel factors that can protect MSCs against an inflammatory environment may enhance the effectiveness of these cells in repairing tissues, such as articular cartilage. In this study, we investigated whether a peptide (P15-1) that binds to hyaluronan (HA), a major component of the extracellular matrix of cartilage, protects bone-marrow-derived MSCs (BMSCs) in an inflammatory environment. The results showed that P15-1 reduced the mRNA levels of catabolic and inflammatory markers in interleukin-1beta (IL-1β)-treated human BMSCs. In addition, P15-1 enhanced the attachment of BMSCs to HA-coated tissue culture dishes and stimulated the chondrogenic differentiation of the multipotential murine C3H/10T1/2 MSC line in a micromass culture. In conclusion, our findings suggest that P15-1 may increase the capacity of BMSCs to repair cartilage via the protection of these cells in an inflammatory environment and the stimulation of their attachment to an HA-containing matrix and chondrogenic differentiation.

We confirm that like decreased FOXA1 expression, decreased PTEN is common in bladder cancer. However, decreased PTEN expression is not associated with a specific morphologic characteristic or gene expression subtype. We show copy number alterations are likely a major factor determining PTEN expression, while FOXA1 expression is relatively independent of copy number. Combined inactivation of Foxa1 and Pten in mice results in the development of bladder cancer with squamous features. Additionally, exposure of these mice to a bladder-specific carcinogen results in rapid development of advanced disease with extensive squamous differentiation.CONCLUSIONS: In summary, through the integration of clinical and animal studies, we identify genetic events which contribute to reduced PTEN and FOXA1 expression, and provide evidence of their contribution to human disease.

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