Proteomic Analyses of the G Protein-Coupled Estrogen Receptor GPER1 Reveal Constitutive Links to Endoplasmic Reticulum, Glycosylation, Trafficking, and Calcium Signaling.

The G protein-coupled estrogen receptor 1 (GPER1) has been proposed to mediate rapid responses to the steroid hormone estrogen. However, despite a strong interest in its potential role in cancer, whether it is indeed activated by estrogen and how this works remain controversial. To provide new tools to address these questions, we set out to determine the interactome of exogenously expressed GPER1. The combination of two orthogonal methods, namely APEX2-mediated proximity labeling and immunoprecipitation followed by mass spectrometry, gave us high-confidence results for 73 novel potential GPER1 interactors. We found that this GPER1 interactome is not affected by estrogen, a result that mirrors the constitutive activity of GPER1 in a functional assay with a Rac1 sensor. We specifically validated several hits highlighted by a gene ontology analysis. We demonstrate that CLPTM1 interacts with GPER1 and that PRKCSH and GANAB, the regulatory and catalytic subunits of α-glucosidase II, respectively, associate with CLPTM1 and potentially indirectly with GPER1. An imbalance in CLPTM1 levels induces nuclear association of GPER1, as does the overexpression of PRKCSH. Moreover, we show that the Ca2+ sensor STIM1 interacts with GPER1 and that upon STIM1 overexpression and depletion of Ca2+ stores, GPER1 becomes more nuclear. Thus, these new GPER1 interactors establish interesting connections with membrane protein maturation, trafficking, and calcium signaling.

A lipid scramblase TMEM41B is involved in the processing and transport of GPI-anchored proteins.

Protein modification by glycosylphosphatidylinositol (GPI) takes place in the endoplasmic reticulum (ER). GPI-anchored proteins (GPI-APs) formed in the ER are transported to the cell surface through the Golgi apparatus. During transport, the GPI-anchor structure is processed. In most cells, an acyl chain modified to the inositol of GPI is removed by a GPI-inositol deacylase, PGAP1, in the ER. Inositol-deacylated GPI-APs become sensitive to bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). We previously reported that GPI-APs are partially resistant to PI-PLC when PGAP1 activity is weakened by the deletion of selenoprotein T (SELT) or cleft lip and palate transmembrane protein 1 (CLPTM1). In this study, we found that the loss of TMEM41B, an ER-localized lipid scramblase, restored PI-PLC sensitivity of GPI-APs in SELT-knockout (KO) and CLPTM1-KO cells. In TMEM41B-KO cells, the transport of GPI-APs as well as transmembrane proteins from the ER to the Golgi was delayed. Furthermore, the turnover of PGAP1, which is mediated by ER-associated degradation, was slowed in TMEM41B-KO cells. Taken together, these findings indicate that inhibition of TMEM41B-dependent lipid scrambling promotes GPI-AP processing in the ER through PGAP1 stabilization and slowed protein trafficking.

Clptm1, a new target in suppressing epileptic seizure by regulating GABAA R-mediated inhibitory synaptic transmission in a PTZ-induced epilepsy model.

Disruption of gamma-amino butyric acid type A receptors (GABAA Rs) synaptic clustering and a decrease in the number of GABAA Rs in the plasma membrane are thought to contribute to alteration of the balance between excitatory and inhibitory neurotransmission, which promotes seizure induction and propagation. The multipass transmembrane protein cleft lip and palate transmembrane protein 1 (Clptm1) controls the forward trafficking of GABAA R, thus decaying miniature inhibitory postsynaptic current (mIPSC) of inhibitory synapses. In this study, using a pentylenetetrazol (PTZ)-induced epilepsy rat model, we found that Clptm1 regulates epileptic seizures by modulating GABAA R-mediated inhibitory synaptic transmission. First, we showed that Clptm1 expression was elevated in the PTZ-induced epileptic rats. Subsequently, we found that downregulation of Clptm1 expression protected against PTZ-induced seizures, which was attributed to an increase in the number of GABAA Rγ2s in the plasma membrane and the amplitude of mIPSC. Taken together, our findings identify a new anti-seizure target that provides a theoretical basis for the development of novel strategies for the prevention and treatment of epilepsy.

Exploring the Relationship between CLPTM1L-MS2 Variants and Susceptibility to Bladder Cancer.

CLPTM1L (Cleft Lip and Palate Transmembrane Protein 1-Like) has previously been implicated in tumorigenesis and drug resistance in cancer. However, the genetic link between CLPTM1L and bladder cancer remains uncertain. In this study, we investigated the genetic association of variable number of tandem repeats (VNTR; minisatellites, MS) regions within CLPTM1L with bladder cancer. We identified four CLPTM1L-MS regions (MS1~MS4) located in intron regions. To evaluate the VNTR polymorphic alleles, we analyzed 441 cancer-free controls and 181 bladder cancer patients. Our analysis revealed a higher frequency of specific repeat sizes within the MS2 region in bladder cancer cases compared to controls. Notably, 25 and 27 repeats were exclusively present in the bladder cancer group. Moreover, rare alleles within the medium-length repeat range (25-29 repeats) were associated with an elevated bladder cancer risk (odds ratio [OR] = 5.78, 95% confidence interval [CI]: 1.49-22.47, p = 0.004). We confirmed that all MS regions followed Mendelian inheritance, and demonstrated that MS2 alleles increased CLPTM1L promoter activity in the UM-UC3 bladder cancer cells through a luciferase assay. Our findings propose the utility of CLPTM1L-MS regions as DNA typing markers, particularly highlighting the potential of middle-length rare alleles within CLPTM1L-MS2 as predictive markers for bladder cancer risk.

Functional characterization of 5p15.33 risk locus in uveal melanoma reveals rs452384 as a functional variant and NKX2.4 as an allele-specific interactor.

The TERT/CLPTM1L risk locus on chromosome 5p15.33 is a pleiotropic cancer risk locus in which multiple independent risk alleles have been identified, across well over ten cancer types. We previously conducted a genome-wide association study in uveal melanoma (UM), which uncovered a role for the TERT/CLPTM1L risk locus in this intraocular tumor and identified multiple highly correlated risk alleles. Aiming to unravel the biological mechanisms in UM of this locus, which contains a domain enriched in active chromatin marks and enhancer elements, we demonstrated the allele-specific enhancer activity of this risk region using reporter assays. In UM, we identified the functional variant rs452384, of which the C risk allele is associated with higher gene expression, increased CLPTM1L expression in UM tumors, and a longer telomere length in peripheral blood mononuclear cells. Electrophoretic mobility shift assays and quantitative mass spectrometry identified NKX2.4 as an rs452384-T-specific binding protein, whereas GATA4 preferentially interacted with rs452384-C. Knockdown of NKX2.4 but not GATA4 resulted in increased TERT and CLPTM1L expression. In summary, the UM risk conferred by the 5p locus is at least partly due to rs452384, for which NKX2.4 presents strong differential binding activity and regulates CLPTM1L and TERT expression. Altogether, our work unraveled some of the complex regulatory mechanisms at the 5p15.33 susceptibility region in UM, and this might also shed light on shared mechanisms with other tumor types affected by this susceptibility region.

Relative Leukocyte Telomere Length and Genetic Variants in Telomere-Related Genes and Serum Levels Role in Age-Related Macular Degeneration.

Telomere shortening is well known to be associated with ageing. Age is the most decisive risk factor for age-related macular degeneration (AMD) development. The older the individual, the higher the AMD risk. For this reason, we aimed to find any associations between telomere length, distribution of genetic variants in telomere-related genes (TERT, TERT-CLPTM1, TRF1, TRF2, and TNKS2), and serum TERF-1 and TERF2 levels on AMD development. METHODS: Our study enrolled 342 patients with AMD and 177 healthy controls. Samples of DNA from peripheral blood leukocytes were extracted by DNA salting-out method. The genotyping of TERT rs2736098, rs401681 in TERT-CLPTM1 locus, TRF1 rs1545827, rs10107605, TNKS2 rs10509637, rs10509639, and TRF2 rs251796 and relative leukocyte telomere length (T/S) measurement were carried out using the real-time polymerase chain reaction method. Serum TERF-1 and TERF2 levels were measured by enzymatic immunoassay (ELISA). RESULTS: We found longer telomeres in early AMD patients compared to the control group. Additionally, we revealed that minor allele C at TRF1 rs10107605 was associated with decreases the odds of both early and exudative AMD. Each minor allele G at TRF2 rs251796 and TRF1 rs1545827 C/T genotype and C/T+T/T genotypes, compared to the C/C genotype, increases the odds of having shorter telomeres. Furthermore, we found elevated TERF1 serum levels in the early AMD group compared to the control group. CONCLUSIONS: In conclusion, these results suggest that relative leukocyte telomere length and genetic variants of TRF1 and TRF2 play a role in AMD development. Additionally, TERF1 is likely to be associated with early AMD.

Genetic Variants at the APOE Locus Predict Cardiometabolic Traits and Metabolic Syndrome: A Taiwan Biobank Study.

Several apolipoprotein genes are located at the APOE locus on chromosome 19q13.32. This study explored the genetic determinants of cardiometabolic traits and metabolic syndrome at the APOE locus in a Taiwanese population. A total of 81,387 Taiwan Biobank (TWB) participants were enrolled to undergo genotype−phenotype analysis using data from the Axiom Genome-Wide CHB arrays. Regional association analysis with conditional analysis revealed lead single-nucleotide variations (SNVs) at the APOE locus: APOE rs7412 and rs429358 for total, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol levels; CLPTM1 rs3786505 and rs11672748 for LDL and HDL cholesterol levels; and APOC1 rs438811 and APOE-APOC1 rs439401 for serum triglyceride levels. Genotype−phenotype association analysis revealed a significant association of rs429358 and rs438811 with metabolic syndrome and of rs7412, rs438811, and rs439401 with serum albumin levels (p < 0.0015). Stepwise regression analysis indicated that CLPTM1 variants were independently associated with LDL and HDL cholesterol levels (p = 3.10 × 10−15 for rs3786505 and p = 1.48 × 10−15 for rs11672748, respectively). APOE rs429358 and APOC1 rs438811 were also independently associated with metabolic syndrome (p = 2.29 × 10−14) and serum albumin levels (p = 3.80 × 10−6), respectively. In conclusion, in addition to APOE variants, CLPTM1 is a novel candidate locus for LDL and HDL cholesterol levels at the APOE gene region in Taiwan. Our data also indicated that APOE and APOC1 variants were independently associated with metabolic syndrome and serum albumin levels, respectively. These results revealed the crucial role of genetic variants at the APOE locus in predicting cardiometabolic traits and metabolic syndrome.

Cumulative Evidence for Relationships Between Multiple Variants in the TERT and CLPTM1L Region and Risk of Cancer and Non-Cancer Disease.

Background: Genetic studies previously reported that variants in TERT-CLPTM1L genes were related to susceptibility of cancer and non-cancer diseases. However, conclusions were not always concordant. Methods: We performed meta-analyses to assess correlations between 23 variants within TERT-CLPTM1L region and susceptibility to 12 cancers and 1 non-cancer disease based on data in 109 papers (involving 139,510 cases and 208,530 controls). Two approaches (false-positive report probability test and Venice criteria) were adopted for assessing the cumulative evidence of significant associations. Current study evaluated the potential role of these variants based on data in Encyclopedia of DNA Elements (ENCODE) Project. Results: Thirteen variants were statistically associated with susceptibility to 11 cancers and 1 non-cancer disease (p < 0.05). Besides, 12 variants with eight cancers and one non-cancer disease were rated as strong evidence (rs2736098, rs401681, and rs402710 in bladder cancer; rs2736100, rs2853691, and rs401681 in esophageal cancer; rs10069690 in gastric cancer; rs2736100 and rs2853676 in glioma; rs2242652, rs2736098, rs2736100, rs2853677, rs31489, rs401681, rs402710, rs465498, and rs4975616 in lung cancer; rs2736100 in idiopathic pulmonary fibrosis and myeloproliferative neoplasms; and rs401681 in pancreatic and skin cancer). According to data from ENCODE and other public databases, 12 variants with strong evidence might fall within putative functional regions. Conclusions: This paper demonstrated that common variants of TERT-CLPTM1L genes were related to susceptibility to bladder, esophageal, gastric, lung, pancreatic, and skin cancer, as well as to glioma, myeloproliferative neoplasms, and idiopathic pulmonary fibrosis, and, besides, the crucial function of the TERT-CLPTM1L region in the genetic predisposition to human diseases is elucidated.

Genome-wide CRISPR screen reveals CLPTM1L as a lipid scramblase required for efficient glycosylphosphatidylinositol biosynthesis.

SignificanceScramblases translocate lipids across the lipid bilayer without consumption of ATP, thereby regulating lipid distributions in cellular membranes. Cytosol-to-lumen translocation across the endoplasmic reticulum (ER) membrane is a common process among lipid glycoconjugates involved in posttranslational protein modifications in eukaryotes. These translocations are thought to be mediated by specific ER-resident scramblases, but the identity of these proteins and the underlying molecular mechanisms have been elusive. Here, we show that CLPTM1L, an integral membrane protein with eight putative transmembrane domains, is the major lipid scramblase involved in efficient glycosylphosphatidylinositol biosynthesis in the ER membrane. Our results validate the long-standing hypothesis that lipid scramblases ensure the efficient translocations of lipid glycoconjugates across the ER membrane for protein glycosylation pathways.

CLPTM1L Is a Novel Putative Oncogene Promoting Tumorigenesis in Oral Squamous Cell Carcinoma.

This study aimed to explore the function of CLPTM1L in oral squamous cell carcinoma and mechanism of tumorigenesis. The expression of CLPTM1L was detected by immunohistochemistry. The localization in cells was detected by immunofluorescence. Cell invasion, proliferation, and migration were detected by transwell, CCK-8 and scratch-wound test. The possible characteristics of CLPTM1L were analysed in TCGA, GO, KEGG and String databases. IHC revealed that the expression of CLPTM1L in 92 cases of OSCC tissues was significantly higher (P < 0.01) than 29 cases of normal oral epithelium tissues. The expression of CLPTM1L was significantly higher in oral squamous cell carcinoma in TCGA database. CLPTM1L expression was not significantly correlated with the patients' clinical parameters. High expression of CLPTM1L was associated with worse prognosis. Cox regression analysis demonstrated that the CLPTM1L expression was the significant risk factor. CLPTM1L was mainly localized in the perinuclear cytoplasm. The vitro studies revealed that the knockdown of CLPTM1L suppressed invasion, proliferation and migration. CLPTM1L related genes were enriched in protein processing in the endoplasmic reticulum, protein folding, endoplasmic reticulum formation, N-glycan biosynthesis, and protein hydroxylation. Highly expressed CLPTM1L may contribute to a poor prognosis and increase invasion, proliferation and migration of oral squamous cell carcinoma. CLPTM1L may play an important role in tumorgenesis and would be a valuable target gene for the treatment of oral squamous cell carcinoma.

Regulation of GABAARs by Transmembrane Accessory Proteins.

The vast majority of fast inhibitory transmission in the brain is mediated by GABA acting on GABAA receptors (GABAARs), which provides inhibitory balance to excitatory drive and controls neuronal output. GABAARs are also effectively targeted by clinically important drugs for treatment in a number of neurological disorders. It has long been hypothesized that function and pharmacology of GABAARs are determined by the channel pore-forming subunits. However, recent studies have provided new dimensions in studying GABAARs due to several transmembrane proteins that interact with GABAARs and modulate their trafficking and function. In this review, we summarize recent findings on these novel GABAAR transmembrane regulators and highlight a potential avenue to develop new GABAAR psychopharmacology by targeting these receptor-associated membrane proteins.

Combination of Clptm1L and TMEM207 Expression as a Robust Prognostic Marker in Oral Squamous Cell Carcinoma.

Overexpression of Cleft Lip and Palate Transmembrane 1-Like (Clptm1L) confers cancer cell survival through the endoplasmic reticulum (ER) stress survival signaling pathway, while TMEM207 impairs the tumor suppressor function of WW domain containing oxidoreductase (WWOX), which sensitizes cancer cells to ER stress-induced apoptosis. In the present study, we examined whether these two ER stress-related proteins, Clptm1L and TMEM207, could be prognostic markers in oral squamous cell carcinoma (OSCC). Immunohistochemical staining using specific antibodies to Clptm1L or TMEM207 revealed that 31 of 89 tissue specimens exhibited concomitant expression of Clptm1L and TMEM207 at the cancer invasion front. A Kaplan-Meier plot of the patient survival curve followed by a log-rank test revealed that the coexpression of Clptm1L and TMEM207 was significantly associated with poor outcome in patients with OSCC (P = 0.00252). Coexpression of Clptm1L and TMEM207 was closely related to lymph node metastasis (P=0.000574). Both univariate and multivariate analyses demonstrated that coexpression of Clptm1L and TMEM207 predicted the poor prognosis of the patients with OSCC. The present study indicated that the double positive Clptm1L and TMEM207 immunoreactivity was closely related to lymph node metastasis with prognostic value in patients with OSCC.

The Prognostic Significance of TERT Locus Polymorphism (rs36115365) in Surgically Resected Non-Small Cell Lung Cancer.

OBJECTIVE: Telomere length is an important factor for the development of non-small cell lung cancer (NSCLC), and current articles focused on telomere associated genes. We studied the clinicopathological and prognostic implications of rs36115365 polymorphism of the TERT-CLPTM1L locus in NSCLC. The association between rs36115365 and telomere length was investigated in 176 NSCLCs. METHODS: DNA was extracted from NSCLC tissues and polymorphism and telomere length were analyzed. RESULTS: The rs36115365 polymorphism showed the following frequencies according to the genotype: G/G in 81.8% of the patients, G/C in 14.2%, and C/C in 4.0%. Average telomere length in the tumor tissues were 3.06-fold longer than telomeres in paired non-tumor tissues (SD=1.87), and telomere length was not significantly different according to rs36115365 (p=0.134). The rs36115365 polymorphism did not have any relationships with clinicopathological characteristics. A poor overall survival result was found in NSCLC with C allele carriers than that with G/G allele (p=0.034). However, disease free survival rate was not different statistically (p=0.938). CONCLUSIONS: These findings suggest that rs36115365 may contribute to the progression of NSCLC.

CLPTM1L induces estrogen receptor ß signaling-mediated radioresistance in non-small cell lung cancer cells.

INTRODUCTION: Radioresistance is a major challenge in lung cancer radiotherapy, and new radiosensitizers are urgently needed. Estrogen receptor ß (ERß) is involved in the progression of non-small cell lung cancer (NSCLC), however, the role of ERß in the response to radiotherapy in lung cancer remains elusive. In the present study, we investigated the mechanism underlying ERß-mediated transcriptional activation and radioresistance of NSCLC cells. METHODS: Quantitative real-time PCR, western blot and immunohistochemistry were used to detect the expression of CLPTM1L, ERß and other target genes. The mechanism of CLPTM1L in modulation of radiosensitivity was investigated by chromatin immunoprecipitation assay, luciferase reporter gene assay, immunofluorescence staining, confocal microscopy, coimmunoprecipitation and GST pull-down assays. The functional role of CLPTM1L was detected by function assays in vitro and in vivo. RESULTS: CLPTM1L expression was negatively correlated with the radiosensitivity of NSCLC cell lines, and irradiation upregulated CLPTM1L in radioresistant (A549) but not in radiosensitive (H460) NSCLC cells. Meanwhile, IR induced the translocation of CLPTM1L from the cytoplasm into the nucleus in NSCLC cells. Moreover, CLPTM1L induced radioresistance in NSCLC cells. iTRAQ-based analysis and cDNA microarray identified irradiation-related genes commonly targeted by CLPTM1L and ERß, and CLPTM1L upregulated ERß-induced genes CDC25A, c-Jun, and BCL2. Mechanistically, CLPTM1L coactivated ERß by directly interacting with ERß through the LXXLL NR (nuclear receptor)-binding motif. Functionally, ERß silencing was sufficient to block CLPTM1L-enhanced radioresistance of NSCLC cells in vitro. CLPTM1L shRNA treatment in combination with irradiation significantly inhibited cancer cell growth in NSCLC xenograft tumors in vivo. CONCLUSIONS: The present results indicate that CLPTM1L acts as a critical coactivator of ERß to promote the transcription of its target genes and induce radioresistance of NSCLC cells, suggesting a new target for radiosensitization in NSCLC therapy. Video Abstract.

Co-exposure to multiple metals, TERT-CLPTM1L variants, and their joint influence on leukocyte telomere length.

OBJECTIVE: Telomere is required for maintaining chromosome stability and genome integrity, while telomere length is sensitive to environmental stressors. We aimed to identify the effects of multiple metals co-exposure as well as their joint effects with TERT-CLPTM1L variants on leukocyte telomere length (LTL). METHODS: This study included 842 workers from a coke-oven plant, of whom plasma concentrations of 23 metals and LTL were determined. Genetic variations in TERT-CLPTM1L were genotyped by using the Global Screening Array. Multipollutant-based statistical methods, including the Bonferroni-correction, backward elimination procedure, and LASSO penalized regression analysis, were used to select the LTL-associated metals. Generalized linear regression models were used to evaluate the joint effects of TERT-CLPTM1L variants with positive metal on LTL. RESULTS: Each 1% increase in plasma concentration of manganese (Mn) was significantly associated with a 0.153% increase in LTL [ß(95%CI) = 0.153(0.075, 0.230), P < 0.001] in single-metal models after Bonferroni-correction. The multiple-metal models and the LASSO penalized regression analysis both indicated Mn as the sole significant predictor for LTL. Furthermore, 5 tagSNPs (rs33954691, rs6554759, rs465498, rs2455393, and rs31489) in TERT-CLPTM1L with high plasma Mn (>4.21 µg/L) showed joint effects on increasing LTL. CONCLUSIONS: Our study revealed the independent and positive association between plasma Mn and LTL when accounting for co-exposure to other metals. This effect can be further enhanced by TERT-CLPTM1L variants. These results may advance our understanding of the complex interplay between genetic and environmental factors on telomere length. Further experimental studies are warranted to elucidate the underlying mechanisms.

Two-stage Bayesian GWAS of 9576 individuals identifies SNP regions that are targeted by miRNAs inversely expressed in Alzheimer's and cancer.

INTRODUCTION: We compared genetic variants between Alzheimer's disease (AD) and two age-related cancers-breast and prostate -to identify single-nucleotide polymorphisms (SNPs) that are associated with inverse comorbidity of AD and cancer. METHODS: Bayesian multinomial regression was used to compare sex-stratified cases (AD and cancer) against controls in a two-stage study. A ±500 KB region around each replicated hit was imputed and analyzed after merging individuals from the two stages. The microRNAs (miRNAs) that target the genes involving these SNPs were analyzed for miRNA family enrichment. RESULTS: We identified 137 variants with inverse odds ratios for AD and cancer located on chromosomes 19, 4, and 5. The mapped miRNAs within the network were enriched for miR-17 and miR-515 families. DISCUSSION: The identified SNPs were rs4298154 (intergenic), within TOMM40/APOE/APOC1, MARK4, CLPTM1, and near the VDAC1/FSTL4 locus. The miRNAs identified in our network have been previously reported to have inverse expression in AD and cancer.

Looking for Novelty in an "Old" Receptor: Recent Advances Toward Our Understanding of GABAARs and Their Implications in Receptor Pharmacology.

Diverse populations of GABAA receptors (GABAARs) throughout the brain mediate fast inhibitory transmission and are modulated by various endogenous ligands and therapeutic drugs. Deficits in GABAAR signaling underlie the pathophysiology behind neurological and neuropsychiatric disorders such as epilepsy, anxiety, and depression. Pharmacological intervention for these disorders relies on several drug classes that target GABAARs, such as benzodiazepines and more recently neurosteroids. It has been widely demonstrated that subunit composition and receptor stoichiometry impact the biophysical and pharmacological properties of GABAARs. However, current GABAAR-targeting drugs have limited subunit selectivity and produce their therapeutic effects concomitantly with undesired side effects. Therefore, there is still a need to develop more selective GABAAR pharmaceuticals, as well as evaluate the potential for developing next-generation drugs that can target accessory proteins associated with native GABAARs. In this review, we briefly discuss the effects of benzodiazepines and neurosteroids on GABAARs, their use as therapeutics, and some of the pitfalls associated with their adverse side effects. We also discuss recent advances toward understanding the structure, function, and pharmacology of GABAARs with a focus on benzodiazepines and neurosteroids, as well as newly identified transmembrane proteins that modulate GABAARs.

Polymorphisms of the TERT-CLPTM1L Gene Are Associated with Pharynx-Larynx Cancer.

Pharynx-larynx cancer is a complex malignant tumor with the sixth-highest morbidity and mortality rate worldwide. The telomerase reverse transcriptase TERT-CLPTM1L gene, located on chromosome 5p15.33, plays a key role in the occurrence and progression of various cancer. The purpose of this hospital-based case-control study of patients in northern China was to explore the association between two single-nucleotide polymorphisms (SNPs) rs401681 in TERT and rs2736100 in CLPTM1L and the risk of head and neck cancer. We collected samples and relative characteristics and then analyzed the relationship between SNPs and pharynx-larynx cancer susceptibility by logistic regression analysis. The results suggested that the male patients carrying CT and CT+CC genotype model of rs401681 was associated with reduced risk of pharynx-larynx cancer compared with the CC genotype (adjusted odds ratios were 0.701 and 0.704, and 95% confidence intervals were 0.495-0.992 and 0.506-0.980; p-values were 0.045 and 0.038, respectively). In addition, we found that subjects with allele-C showed a relatively low risk of pharyngeal cancer when smoking exposure history was obtained. But the limitation is that in the future we need to further investigate about the exact functional effect of these two variant genes and a larger scale sample. Overall, in this research, our results show that the TERT-CLPTM1L gene could be a meaningful biomarker for pharynx-larynx cancer susceptibility.

Pathobiological role of cleft palate transmembrane protein 1 family proteins in oral squamous cell carcinoma.

PURPOSE: Cleft palate transmembrane protein 1 (Clptm1) and its paralog protein, Cisplatin resistance-related protein 9 (CRR9) constitute a highly conserved protein family, from Caenorhabditis elegans to Homo sapiens. In the present study, we examined the clinicopathological and biological significance of Clptm1 and CRR9 expression in oral squamous cell carcinoma (OSCC). METHODS: Ninety-eight OSCC tissue specimens were immunohistochemically stained with specific antibodies to Clptm1 and CRR9. The immunoreactivity of Clptm1 and CRR9 was then correlated with clinicopathological factors, including the prognosis of patients. siRNA-mediated gene silencing of CRR9 followed by cell proliferation, Matrigel invasion, anoikis assay, and gelatin zymography were performed using cultured OSCC cells. Subsequently, immunohistochemical examination including double staining was performed to determine the correlation between CRR9 and Bcl-xL expression in OSCC cells. RESULTS: Non-tumorous oral squamous cells exhibited vague, weak, or little cytoplasmic staining with anti-Clptm1 and CRR9 antibodies. By contrast, robust Clptm1 and CRR9 immunoreactivity was found at the cancer invasion front in 55 and 54 of the 98 OSCC tissue specimens, respectively. Notably, CRR9 immunoreactivity was associated with more than 5 mm of depth of invasion, poor prognosis of the patients, and smoking habits (P < 0.05). siRNA-mediated gene silencing of CRR9 did not alter the cell proliferation but decreased Matrigel invasion and impaired anoikis resistance in cultured Ca9-22 and SAS cells. CRR9 and anti-apoptotic Bcl-xL expression levels were correlated in pT1 OSCC tissue specimens. CONCLUSIONS: Clptm1 and CRR9 were overexpressed in many OSCC tissues. In particular, CRR9 expression may promote tumor development and have a significant poor prognostic value in OSCC, possibly through conferring invasion ability and resistance to apoptotic stimuli possibly related to Bcl-xL expression. CRR9 could be a novel molecular target for patients with OSCC.

CLPTM1L/CRR9 ectodomain interaction with GRP78 at the cell surface signals for survival and chemoresistance upon ER stress in pancreatic adenocarcinoma cells.

Altered regulation of endoplasmic reticulum (ER) homeostasis has been implicated in many cancers and has recently become a therapeutic and chemosensitization target of interest. We have identified Cleft Lip and Palate Transmembrane 1-Like (CLPTM1L)/Cisplatin Resistance Related Protein 9 (CRR9) as an ER stress related mediator of cytoprotection in pancreatic cancer. We recently demonstrated that CLPTM1L is highly expressed in pancreatic ductal adenocarcinoma and associated with poor outcome. Furthermore, we have discovered that CLPTM1L interacts with phosphoinositol-3-kinase-alpha at the tumor cell surface and causes up-regulation of Bcl-xL and pAkt mediated survival signaling. Here, we demonstrate surface relocalization and survival signaling by CLPTM1L triggered by endoplasmic reticular (ER) stress. We demonstrate the interaction of CLPTM1L with the central ER stress survival mediator, Glucose Regulated Protein 78 (GRP78)/Binding Immunoglobulin Protein (BiP) and PI3K-alpha /p110α. This interaction and surface relocalization of CLPTM1L and GRP78 is induced by ER stress, including that caused by treatment with gemcitabine. We demonstrate that the extracellular loop of CLPTM1L is required for gemcitabine resistance and interaction with GRP78. This interaction and the chemoresistance effect conferred by this pathway is targetable with our recently developed inhibitory CLPTM1L antibodies, which may represent novel modalities of chemosensitization and treatment of pancreatic adenocarcinoma. Anchorage independent growth, GRP78-mediated chemoresistance, and Akt phosphorylation were abrogated by inhibition of CLPTM1L. These findings demonstrate a novel and potentially targetable mechanism of cytoprotection and chemoresistance in pancreatic tumors.