SUMMARY Vesicular acidification and trafficking are associated with various cellular processes. However, their pathologic relevance to cancer remains elusive. We identified transmembrane protein 9 (TMEM9) as a vesicular acidification regulator. TMEM9 is highly upregulated in colorectal cancer (CRC). Proteomic and biochemical analyses show that TMEM9 binds to and facilitates assembly of v-ATPase, a vacuolar proton pump, resulting in enhanced vesicular acidification and trafficking. TMEM9-v-ATPase hyperactivates Wnt/β-catenin signaling via lysosomal degradation of APC. Moreover, TMEM9 transactivated by β-catenin functions as a positive feedback regulator of Wnt signaling in CRC. Genetic ablation of TMEM9 inhibits CRC cell proliferation in vitro, ex vivo, and in vivo mouse models. Moreover, administration of v-ATPase inhibitors suppresses intestinal tumorigenesis of APC mouse models and human patient-derived xenografts. Our results reveal the unexpected roles of TMEM9-controlled vesicular acidification in hyperactivating Wnt/β-catenin signaling through APC degradation, and propose the blockade of TMEM9-v-ATPase as a viable option for CRC treatment.

BackgroundGlioblastoma (GBM) is one of the most lethal tumors with a poor prognosis. Its inevitable recurrence is frequently explained by the presence of cancer stem cells. We aimed to show that human GBM cells with stemness features are more sensitive to natural killer (NK) cells than GBM cells without stemness characteristics.MethodsNatural killer cell cytotoxicity was measured using flow cytometry in neurosphere-forming U87 GBM cells cultured with neurobasal media (NBE condition) and compared with that in serum-cultured U87 GBM cells (serum condition). Cytotoxicity was examined after addition of blocking NKG2D monoclonal antibodies. The expression profile of NK ligands of NK cells were investigated by reverse transcription polymerase chain reaction and western blot analysis in the U87 GBM cells in both conditions.ResultsNBE U87 cells showed higher cytotoxicity to NK cells than serum U87 cells did (55 vs 35% at an effector to target cell ratio of 5:1). The increased cytotoxicity was diminished in NBE U87 cells by a larger gap than in serum U87 cells by adding NKG2D blocking antibodies. Of the NKG2D ligands, the expression of ULBP1 and ULBP3 was relatively increased in NBE U87 cells compared to serum U87 cells.ConclusionsU87 GBM cells with stemness features demonstrate increased cytotoxicity to NK cells in association with altered NKG2D ligand expression of NK cell activating receptor. Applying immune modulation to GBM treatment may be a promising adjuvant therapy in patients with intractable GBM.

The contractile properties of single muscle fibers reflect the functional status of muscle at the cellular level and have not been described in amyotrophic lateral sclerosis (ALS). Chemically skinned single muscle fibers (n = 173), obtained by needle biopsy from six men with ALS, were activated with Ca(2+), allowing maximal force measurements and specific force (SF) estimates. Maximum unloaded shortening velocity (V(o)) was determined using the slack test. The results were compared with muscle from healthy controls. Markers of disease progression included rate of change of ALS functional rating scale score, rate of change of forced vital capacity, and disease duration. Compared with controls, ALS patients had decreased whole muscle SF (measured by a combination of computerized tomography and isokinetic testing) but normal single fiber SF. The V(o) was greater for type I fibers in ALS. Patients with slower disease progression had increased single fiber size and a high percentage of hybrid fibers (expressing multiple myosin heavy chain isoforms). A needle biopsy obtained at the time of ALS diagnosis may assist with predicting rate of disease progression.

Reduced succinate dehydrogenase (SDH) activity resulting in adverse succinate accumulation was previously considered relevant only in 0.05 to 0.5% of kidney cancers associated with germline SDH mutations. Here, we sought to examine a broader role for SDH loss in kidney cancer pathogenesis/progression. We report that underexpression of SDH subunits resulting in accumulation of oncogenic succinate is a common feature in clear cell renal cell carcinoma (ccRCC) (∼80% of all kidney cancers), with a marked adverse impact on survival in ccRCC patients (n = 516). We show that SDH down-regulation is a critical brake in the TCA cycle during ccRCC pathogenesis and progression. In exploring mechanisms of SDH down-regulation in ccRCC, we report that Von Hippel-Lindau loss-induced hypoxia-inducible factor–dependent up-regulation of miR-210 causes direct inhibition of the SDHD transcript. Moreover, shallow deletion of SDHB occurs in ∼20% of ccRCC. We then demonstrate that SDH loss-induced succinate accumulation contributes to adverse loss of 5-hydroxymethylcytosine, gain of 5-methylcytosine, and enhanced invasiveness in ccRCC via inhibition of ten-eleven translocation (TET)-2 activity. Intriguingly, binding affinity between the catalytic domain of recombinant TET-2 and succinate was found to be very low, suggesting that the mechanism of succinate-induced attenuation of TET-2 activity is likely via product inhibition rather than competitive inhibition. Finally, exogenous ascorbic acid, a TET-activating demethylating agent, led to reversal of the above oncogenic effects of succinate in ccRCC cells. Collectively, our study demonstrates that functional SDH deficiency is a common adverse feature of ccRCC and not just limited to the kidney cancers associated with germline SDH mutations.