JMJD5, a Jumonji C domain-containing dioxygenase, is important for embryonic development and cancer growth. Here, we show that JMJD5 is up-regulated by hypoxia and is crucial for hypoxiainduced cell proliferation. JMJD5 interacts directly with pyruvate kinase muscle isozyme (PKM)2 to modulate metabolic flux in cancer cells. The JMJD5-PKM2 interaction resides at the intersubunit interface region of PKM2, which hinders PKM2 tetramerization and blocks pyruvate kinase activity. This interaction also influences translocation of PKM2 into the nucleus and promotes hypoxiainducible factor (HIF)-1α-mediated transactivation. JMJD5 knockdown inhibits the transcription of the PKM2-HIF-1α target genes involved in glucose metabolism, resulting in a reduction of glucose uptake and lactate secretion in cancer cells. JMJD5, along with PKM2 and HIF-1α, is recruited to the hypoxia response element site in the lactate dehydrogenase A and PKM2 loci and mediates the recruitment of the latter two proteins. Our data uncover a mechanism whereby PKM2 can be regulated by factor-bindinginduced homo/heterooligomeric restructuring, paving the way to cell metabolic reprogram.Warburg effect | aerobic glycolysis | breast cancer | cancer metabolism J MJD5 is a Jumonji C domain-containing dioxygenase shown to be involved in lysine demethylation (1-3) and hydroxylation functions (4). Although the exact cellular substrates and functions of JMJD5 remain unclear, JMJD5 was shown to positively regulate cyclin A1 but negatively regulate p53 and p21 (1-3). Knockdown of JMJD5 in Michigan Cancer Foundation (MCF)-7 cells inhibits cell proliferation (1), and JMJD5 −/− embryos showed severe growth retardation, resulting in embryonic lethality at the midgestation stage (3). These data, together with its general overexpression in tumor tissues, implicate a role of JMJD5 in carcinogenesis. In this paper, we define a role of JMJD5 in regulating tumor metabolism under normoxic and hypoxic conditions through its interaction with pyruvate kinase muscle isozyme (PKM)2.One of the hallmarks of cancer cells is their altered metabolism, referred to as aerobic glycolysis, or the Warburg effect (5). This generally involves an increased uptake of glucose, use of intracellular glucose to pyruvate via glycolysis, and the conversion into lactate in the presence of sufficient oxygen. Along this metabolic flux, PKM1 or its spliced variant, PKM2, which dephosphorylates phosphoenolpyruvate (PEP) into pyruvate, the last step of glycolysis, is an important signal integrator whose activities determine the cytosolic level of pyruvate, thereby affecting subsequent metabolic flow to lactate, tricarboxylic acid cycle or biosynthetic pathway (6). Enzymatically, PKM2, an embryonic isoform found abundantly in tumor cells, is less active than PKM1, which allows the accumulation of glycolytic intermediates and diversion into biosynthetic pathways, demanded by rapid-proliferating cells.As a pivotal regulator of tumor metabolism, PKM2's activity is further modulated by allosteric regulation vi...

Photodynamic therapy (PDT) is a kind of photochemo-therapeutic treatment that exerts its effect mainly through the induction of cell death. Distinct types of cell death may be elicited by different PDT regimes. In this study, the mechanisms involved in the death of human epidermoid carcinoma A431 cells triggered by PDT with Photofrin (a clinically approved photosensitizer) were characterized. Photofrin distributes dynamically in A431 cells; the plasma membranes and Golgi complex are the main target sites of Photofrin after a brief (3 h) and prolonged (24 h) incubation, respectively. Cells with differentially localized Photofrin displayed distinct death phenotypes in response to PDT. The effects of PDT on cells with plasma membrane-localized Photofrin were further studied in details. Cells stopped proliferating post PDT at Photofrin dose >7 micro g/ml, and at higher dose (28 micro g/ml) plasma membrane disruption and cell swelling were observed immediately after PDT. Dramatic alterations of several important signaling events were detected in A431 cells post Photofrin-PDT, including (i) immediate formation of reactive oxygen species (ROS), (ii) rapid activation of c-Jun N-terminal kinase, (iii) delayed activation of caspase-3 and cleavage of polyADP-ribose polymerase and p21-activated kinase 2, and (iv) loss of mitochondrial membrane potential. Intriguingly, the characteristics of typical apoptosis such as phosphatidylserine externalization and DNA fragmentation were not detected in the cell death process caused by this PDT regime. In conclusion, our results show that when plasma membranes are the main targets, Photofrin-PDT can lead to instant ROS formation and subsequent activation of downstream signaling events similar to those elicited by many apoptotic stimuli, but the damage of plasma membranes renders the death phenotype more necrosis like.

MYO18A is found as a novel PAK2 binding partner via βPIX/GIT1. MYO18A-depleted cells showed dramatic changes in shape, actin stress fiber and membrane ruffle formation, and displayed increases in the number and size of focal adhesions and a decrease in cell migration, suggesting an important role of MYO18A in regulating epithelial cell migration.

The PAK2/βPIX/GIT1 (p21-activated kinase 2/PAK-interacting exchange factor-β/G protein-coupled receptor kinase-interactor 1) complex has been shown to distribute to both membrane ruffles and focal adhesions of cells, where it plays an important role in regulating focal adhesion turnover. However, the detailed mechanism underlying this regulation is largely unknown. We previously reported that MYO18Aα interacts via its carboxyl terminus with the PAK2/βPIX/GIT1 complex through direct binding to βPIX, and that knockdown of MYO18Aα in epithelial cells causes accumulation of the complex in focal adhesions and decreased cell migration ability (Hsu et al., 2010). The current study characterized the detailed MYO18Aα-βPIX interaction mechanism and the biological significance of this interaction. We found that deletion of the carboxyl-terminal globular domain of MYO18Aα profoundly altered the cellular localization of βPIX and inhibited cell migration. βPIX interacts through its most carboxyl-terminus, PAWDETNL (639-646), with MYO18Aα and partially colocalized with MYO18Aα in membrane ruffles of cells, whereas βPIX(1-638), a mutant with deletion of PAWDETNL, accumulated in focal adhesions. Both focal adhesion numbers and area in βPIX(1-638)-expressing cells were greater than those in cells expressing wild-type βPIX(FL). Further experiments using deletion mutants of MYO18A and βPIX showed that disruption of MYO18A-βPIX interaction not only impaired cell motility but also decreased Rac1 activity. Collectively, our data unravel the interaction regions between MYO18A and βPIX and provide evidence for the critical role of this interaction in regulating cellular localization of βPIX, Rac1 activity, and adhesion and migration in epithelial cells.

BACKGROUND A previous comparative tissue proteomics study by the authors of the current study led to the identification of caldesmon (CaD) as one of the proteins associated with cervical metastasis of oral cavity squamous cell carcinoma (OSCC). In the current investigation, the authors focused on the potential functions of CaD in patients with OSCC. METHODS CaD expression was examined in tissue samples from 155 patients using immunohistochemical analysis. The expression of CaD variants was determined by Western blot analysis and reverse transcriptase‐polymerase chain reaction. In addition, the specific effects of CaD gene overexpression and silence were determined in OSCC cell lines. RESULTS CaD expression was found to be significantly higher in tumor cells from metastatic lymph nodes compared with primary tumor cells, and was nearly absent in normal oral epithelia. Higher CaD expression was found to be correlated with positive N classification, poor differentiation, perineural invasion, and tumor depth (P = .001, P = .029, P = .001, and P = .031, respectively). In survival analyses, OSCC patients with higher CaD expression were found to have poorer prognosis with regard to disease‐specific survival and disease‐free survival (P = .003 and P = .014, respectively). Multivariate analyses further indicated that higher CaD expression was an independent predictor of disease‐specific survival (P = .043). Serum CaD levels were found to be significantly higher in patients with OSCC, but this finding was not associated with clinicopathological manifestations. Data obtained from in vitro suppression, rescue, and overexpression of CaD in OEC‐M1 cells indicated that CaD promotes migration and invasive processes in OSCC cells. CONCLUSIONS The findings of the current study collectively suggest that the low‐molecular‐weight CaD expression in OSCC tumors is associated with tumor metastasis and patient survival. Cancer 2013;119:4003–4011. © 2013 American Cancer Society.