Breast cancer is the most common cancer in women among nearly every racial and ethnic group. About one in eight women in the US will get breast cancer in her lifetime. Sigma 2 receptor has long been implicated in breast carcinogenesis and compounds binding to this receptor have been developed as imaging agents for breast cancer, but its molecular identity had been elusive until 2017 when TMEM97 was identified as sigma 2 receptor. It is highly important to determine whether the biological functions of TMEM97 protein defined in previous studies are overlapping with those linked to sigma 2 receptor. In this study, we found that TMEM97 is highly expressed in ER positive breast tumors and its expression levels are associated with poor overall survival rate of breast cancer patients, and its expression patten is strongly correlated with ER and PR but not with HER2 status. Breast cancer cells with TMEM97 overexpression showed growth advantage over the control cells both in nutrition sufficient and starvation conditions. The differences were more pronounced under estrogen depleted conditions. In breast cancer MCF7 and T47D cells, TMEM97/sigma 2 receptor could regulate ERα transcriptional activities, and also regulate mTOR/S6K1 signaling pathways. The increased level of active, phosphorylated ERα, and the increased resistance to Tamoxifen treatment could be blocked by a mTOR inhibitor. TMEM97 in ER positive breast cancer cells could retard and delay the estradiol caused ERα turnover because of the accelerated the ERα protein synthesis offsetting the degradation caused by estradiol. TMEM97 increases the transactivated ERα, specifically for the nuclear soluble and chromatin bounded ERα. These observations suggest that TMEM97/sigma 2 receptor participates in breast tumor cell growth driven by estrogen signaling and further regulates the transcriptional activities of ERα through modulating ERα binding to estradiol and subsequent nuclear localization. Further, increased TMEM97 expression renders breast cancer cells with increased resistance toward endocrine therapeutics such as tamoxifen, which make TMEM97 a valid target of intervention to modulate ER activities to reduce resistance toward endocrine therapy.

Alcohol is abundantly consumed by society and general anesthetics are used everyday in operating suites throughout the world, yet the sites and mechanisms of action for these drugs are not completely understood. Glycine receptors (GlyRs) are pentameric ion channels expressed throughout the brain and spinal cord and have become increasingly popular targets in the study of alcohol action. Each GlyR subunit is composed of four alpha helical transmembrane segments (TM1-4), and although amino acids involved with alcohol action have been previously identified in TM1-4, the orientation of each of these residues with respect to a putative alcohol/anesthetic binding cavity remains controversial. In order to better characterize this binding cavity within the GlyR, we conducted a series of experiments using cysteine mutagenesis and biochemical cross-linking. In Aim 1, the participation of TM1 with TM3 in a common alcohol/anesthetic binding cavity was further investigated. We used two-electrode voltage clamp electrophysiology in Xenopus oocytes to demonstrate the ability of A288 in TM3 to form cross-links with I229 in TM1, which reduced the ability of both alcohol and anesthetics to modulate GlyR function. Aim 2 investigated whether TM3 could also participate in a binding cavity with TM4. We have shown that residues in TM4 are able to form cross-links with A288 in TM3, and found that cross-linking between TM3 and those residues in TM4 also reduced the ability of alcohol and anesthetics to enhance GlyR function. Aim 3 determined whether these cross-links are formed between residues within the same subunit (intra-subunit) or between subunits (inter-subunit), and ultimately whether these residues participate in a common alcohol/anesthetic binding cavity within or between GlyR subunits. GlyR protein, which measures about 50 kDa, was extracted from oocytes injected with the cysteine mutants, and immunoblotting was used with a GlyR-specific antibody to subsequently help quantify band ratios between cross-linked and uncross-linked conditions. We found an increase in the 100:50 kDa band ratio for the TM1-3 mutant only, but not TM3-4 mutant or the wild-type, which suggests TM1-3 may participate in an alcohol binding cavity between GlyR subunits while TM3-4 may contribute to a binding cavity within a subunit. / text

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