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INTRODUCTION AND OBJECTIVES: The metabolism of cancer cells is fundamentally different from that of their normal counterparts. The M2 isoenzyme of pyruvate kinase (PKM2), expressed primarily during embryogenesis, is switched back on and exerts two major effects during oncogenesis: (i) slowing down glycolysis thus shunting glycolytic intermediates for anabolic process necessary for cell proliferation; and (ii) interacting with transcriptional factors thus upregulating growth-promoting genes. The role of PKM2 in bladder cancer formation and progression is presently unknown. METHODS: Human specimens and cultured cells from normal urothelia, low-gradenon-invasive and high-grademuscle-invasive bladder cancerwereassessed forPKM2expressionby immunohistochemistryand immunoblotting. Selected cancer cell lines over-expressing PKM2 were treated with shikonin, a naphthoquinone inhibitor of PKM2 found in medicinal plants, alone or in combination with rapamycin, an mTOR inhibitor, and their effects on cell proliferation, metabolism, apoptotic, autophagic and unfolded protein responses were determined using biochemical and molecular approaches. Target-specific effects of shikonin were verified by transfection with PKM2-specifc small interfering RNA (siRNA). RESULTS: Compared to normal urothelia, both low-grade noninvasive and high-grade muscle-invasive bladder cancer markedly over-express PKM2. Accompanying this is the hyper-phosphorylation of tyrosine residue at codon Y105 and nuclear translocation of PKM2. Cultured bladder cancer cell lines including UroTsa, RT4, RT112, J82, T24 and UMUC3 also express significantly more PKM2 than primarycultured urothelial cells. Exposure of RT112 and T24 to shikonin results in a dose-dependent inhibition of proliferation, reduced oxygen consumption and marked upregulation of signals indicative of apoptosis, autophage and unfolded protein response. A greater inhibition of cell proliferation is observed when shikonin is combined with rapamycin, suggesting a synergism between PKM2 and mTOR inhibition. Enforced expression of siRNA of PKM2 produces a similar inhibitory effect on cell line proliferation, establishing target-specific effects of shikonin. CONCLUSIONS: These data demonstrate for the first time an important role of PKM2 overexpression in bladder cancer formation and progression, and suggest a potentially unconventional way to therapeutically inhibit bladder cancer via specific alteration of tumor-promoting metabolism.

INTRODUCTION AND OBJECTIVES: Recent whole-genome/exome sequencing and multiplatform analysis of human bladder cancer led to two key discoveries: (i) most muscle invasive bladder cancer (MIBC) carry molecular alterations that activate the receptor tyrosine/Ras/PI3K pathway and those that inactivate the p53 pathway; and (ii) MIBC can be subclassified into luminal (luminal and p53-like) and basal subtypes with different molecular signatures, responses to therapy and prognosis. The exact relationship between the molecular alterations and the subtypes, however, still awaits experimental validation. METHODS: Mutation-activated Ras and p53 deletion was achieved in urothelium of transgenic mice and knockout mice, respectively. The two types of mice were further inter-crossed to yield mice both expressing activated Ras and lacking p53 in urothelium. Histopathology, immunohistochemistry, Western blotting, Real-time PCR, cell-cycle distribution and transcriptome profiling were performed to evaluate the phenotypic and molecular changes. Effects on cell growth, motility and invasion were verified in cultured RT4 cells by transfection of activated Ras and/or knockdown of p53. RESULTS: Ras activation in urothelium resulted in persistent hyperplasia which did not progress to tumors. This was due primarily to a markedly induced failsafe mechanism in the p53 pathway. Although p53 deletion in urothelium led to a normal morphology, its deletion in urothelium constitutively expressing the activated Ras caused carcinoma in situ and MIBC in 60% of the mice by 14 months of age. The invasive lesions had marked increase of transcriptional factors driving the epithelial-mesenchymal transition, mesenchymal markers and extracellular matrix components. The invasive lesions also expressed multiple markers characteristic of cancer progenitor cells (CD44, keratins 14 & 5) and squamous differentiation (keratin 1, Trim29). These features strongly resemble those of the basal subtype MIBC recently classified in humans. Finally, transfection of RT4 cells, derived from a patient’s low-grade papillary tumor, with both activated Ras and shRNA of p53, but not either alone, converted these noninvasive cells into highly invasive ones. CONCLUSIONS: Our data strongly suggest that the combinatorial molecular alterations of both RTK/Ras/PI3K and p53 pathways underlie the basal subtype MIBC and that signal alterations in these two pathways may be used for improved diagnosis, prognosis prediction and therapeutic inhibition.

INTRODUCTION AND OBJECTIVES: Phosphatase and Tensin Homology (PTEN) antagonizes PI3-kinase/AKT and this activity relies on PTEN-membrane interaction. While loss of PTEN, along with p53 defects, triggers muscle-invasive bladder cancer (BC) in genetically engineered mice, PTEN deletion or mutation in human BC is rare, suggesting that PTEN is inactivated via a different mechanism. Hyperphosphorylation of PTEN’s C-terminus (PTEN-Cp) is thought to dissociate PTEN from the membrane, thus functionally inactivating PTEN. This study investigates the prevalence of PTEN-Cp and its effects on tumor suppression in BC cells. METHODS: Ten cell lines representing different grades/stages of human BC was assessed for PTEN-Cp using site-specific antibodies. cDNAs encoding (i) wild-type PTEN, (ii) mutated PTEN devoid of C-terminal phosphorylation sites and (iii) truncated PTEN lacking the C-terminus were transfected into UMUC3 and J82 BC cell lines that lack the PTEN gene. PTEN-Cp status, PI3K/AKT activation and BC cell proliferation were assessed in vitro and in vivo using xenograft models. PTEN-Cp was also assessed using Western blotting of freshly preserved human BC tissues. RESULTS: Prominent PTEN-Cp was observed in all BC cell lines except UMUC3 and J82 which lack the PTEN gene. Transfection of wild-type PTEN into UMUC3 and J82 inhibited cell growth, albeit only at serum-free or low-serum (2%) conditions. High-serum (10%) caused PTEN to undergo extensive PTEN-Cp, increased AKT activation and cell proliferation. Mutated PTEN devoid of C-terminal phosphorylation sites and truncated PTEN lacking the C-terminus exerted much greater inhibitory effects on AKT and cell growth of UMUC3 and J82. These effects were reproducible when the transfected cells were transplanted subcutaneously into xenograft models. Finally, surveying a cohort of fresh human BC tissues revealed that a great majority of lowgrade non-invasive and high-grade invasive BC expressed PTEN and whenever PTEN was expressed it was highly phosphorylated at the C-terminus. CONCLUSIONS: Our studies provide the first evidence demonstrating that hyper-phosphorylation at the C-terminus is a major mechanism of PTEN inactivation in human BC, that PTEN phosphorylation may serve as a useful biomarker for BC progression, and that the tail-less PTEN is a much more potent tumor suppressor than the fulllength PTEN when used in molecular therapy.