Probes for MTOR

Mammalian target of rapamycin (mTOR) regulates various fundamental cellular events including cell proliferation, protein synthesis, metabolism, apoptosis, and autophagy. Tumor suppressive miR-99b-5p has been implicated in regulating PI3K/AKT/mTOR signaling in a variety of types of cancer. Our previous study suggested the reciprocal miR-99b-5p/MTOR (downregulated/upregulated) pairing as a key microRNA-mRNA regulatory component involved in the prostate cancer (PCa) disparities. In this study, we further validated the expression profiles of mTOR and miR-99b-5p in the PCa, colon, breast, and lung cancer specimens and cell lines. The immunohistochemistry (IHC), immunofluorescence, Western blot, and RT-qPCR assays have confirmed that mTOR is upregulated while miR-99b-5p is downregulated in different patient cohorts and a panel of cancer cell lines. Intriguingly, elevated nuclear mTOR expression was observed in African American PCa and other advanced cancers. Transfection of the miR-99b-5p mimic resulted in a significant reduction in nuclear mTOR and androgen receptor (AR), while a slight/moderate to no decrease in cytoplasmic mTOR and AR in PCa and other cancer cells, suggesting that miR-99b-5p inhibits mTOR and AR expression and their nuclear translocation. Moreover, overexpression of miR-99b-5p targets/inhibits AR-mTOR axis, subsequently initiating cell apoptosis and sensitizing docetaxel-induced cytotoxicity in various cancers. In conclusion, our data suggest that reciprocal miR-99b-5p/nuclear mTOR pairing may be a more precise diagnostic/prognostic biomarker for aggressive PCa, than miR-99b-5p/MTOR pairing or mTOR alone. Targeting the AR-mTOR axis using miR-99b-5p has also been suggested as a novel therapeutic strategy to induce apoptosis and overcome chemoresistance in aggressive PCa.

Pachyonychia congenita (PC) is a rare keratinizing disorder characterized by painful palmoplantar keratoderma (PPK) for which there is no standard current treatment. PC is caused by dominant mutations in keratin 6A, 6B, 6C, 16, and 17 genes involved in stress, wound healing, and epidermal barrier formation. Mechanisms leading to pain and PPK in PC remain elusive. Here, we show overexpression of EGFR ligands epiregulin and TGF-α as well as HER1-EGFR and HER2 in the upper spinous layers of PC lesions. EGFR activation was confirmed by upregulated MAPK/ERK and mTOR signaling. Abnormal late terminal keratinization was associated with elevated transglutaminase-1 (TG1) activity. Additionally, the Ca2+ permeable channel TRPV3 was significantly increased in PC-lesional skin suggesting a predominant role of the TRPV3/EGFR signaling complex in PC. We hypothesized that this complex contributes to promoting TG1 activity and induces the expression and shedding of EGFR ligands. To counteract this biological cascade, we treated 3 PC patients with oral erlotinib for 6 to 8 months. The treatment was well tolerated and led to an early, drastic, and sustained reduction of neuropathic pain with a major improvement of quality-of-life. Our study provides evidence that targeted pharmacological inhibition of EGFR is an effective strategy in PC.