Stomatin-like protein 2: a link between T cell activation and mitochondrial function (35.19)

Hepatocellular carcinoma (HCC) treatment remains challenging due to the prevalence of metastasis and chemotherapy resistance. Mitochondrial stomatin-like protein 2 (STOML2), which is upregulated in various solid tumors, is associated with a poor prognosis; however, its biological function and molecular mechanism in HCC remain unclear. The present study aimed to elucidate the oncogenic mechanism of STOML2 in HCC and to explore its potential as a therapeutic target. Firstly, STOML2 expression in HCC and matched normal liver tissues was analyzed. In addition, STOML2-knockdown (HCCLM3-short hairpin RNA-STOML2) and -overexpression (Huh7-STOML2) cell models were established. Wound healing, Cell Counting Kit-8 and Transwell assays, and flow cytometry were performed to assess cell proliferation, invasion, migration and apoptosis in vitro. Furthermore, the biological function of STOML2 was confirmed in vivo. Co-immunoprecipitation (co-IP) and immunofluorescence staining were conducted to validate the interaction of STOML2 with prohibitin (PHB) following the prediction of binding partners. Downstream pathways regulated by STOML2 were identified using western blotting and were further investigated using the RAF1 inhibitor sorafenib. The present study revealed that STOML2 expression was significantly upregulated in HCC tissues and metastatic lesions, and was associated with poor patient prognosis. The in vitro experiments showed that STOML2 overexpression promoted proliferation, invasion, migration and autophagy, while inhibiting apoptosis in Huh7 cells. Conversely, STOML2 knockdown reversed these phenotypic changes. Furthermore, co-IP confirmed the direct interaction between STOML2 and PHB, which activated the RAF/MEK/ERK signaling pathway. The in vivo experiments further confirmed that STOML2 overexpression significantly accelerated tumor growth, whereas STOML2 or PHB knockdown inhibited tumor progression. In addition, sorafenib treatment suppressed STOML2-mediated cell migration and the expression of autophagy-related proteins by blocking the MAPK pathway. These findings elucidated the molecular mechanism by which STOML2 promotes the malignant progression of HCC and demonstrated that targeted inhibition of the PHB-MAPK pathway may reverse the pro-tumorigenic effects of STOML2. STOML2 may serve as both a prognostic biomarker and a therapeutic target in HCC. The current study provides a theoretical foundation for individualized treatment in patients with HCC and high STOML2 expression.

Stomatin-like protein 2 (STOML2), a member of the stomatin, has been reported to be upregulated in several human cancers. However, its role and clinical significance in gastric adenocarcinoma remains unclear to date. The purpose of this retrospective study was to explore whether there was a correlation between the expression of STOML2 by immunohistochemistry and the clinical outcome of a large group of patients with gastric adenocarcinoma. In this retrospective study, we performed immunohistochemistry to evaluation of STOML2 expression in a large panel of gastric adenocarcinoma samples. The receiver operating characteristic method was used to define the STOML2 immunoreactivity score cutoff value. The clinical/prognostic significance of STOML2 expression was analyzed statistically. Kaplan-Meier analysis was used to compare the postoperative survival between groups. STOML2 was overexpressed in gastric cancer compared with paracancerous normal mucosa. Increased STOML2 expression was associated with higher histologic grade (P = 0.047), T category (P < 0.001), and N category (P = 0.01). Patients with high expression of STOML2 demonstrated shortened overall survival compared with those with low expression of STOML2 (median of 38.9 vs. 64.0 months, P < 0.001). Furthermore, STOML2 expression could stratify patients survival in stage N0 (P < 0.001). Multivariate analysis showed that the level of STOML2 expression was an independent prognostic factor in gastric adenocarcinoma (RR = 1.920, P = 0.001). Increased expression of STOML2 suggests unfavorable prognosis for gastric adenocarcinoma patients. Further studies are warranted.

Objective Asthma is a chronic inflammatory airway disease characterized by airway inflammation and remodeling. Abnormal proliferation and migration of human airway smooth muscle cells (HASMCs), induced by platelet-derived growth factor BB (PDGF-BB), are associated with the occurrence and progression of asthma. In this study, we aim to investigate the expression and molecular mechanisms of stomatin-like protein-2 (STOML2) in airway remodeling in asthma. Methods PDGF-BB-stimulated HASMCs were used as the asthma cell model. STOML2 expression levels in the serum of patients with asthma and healthy controls were measured using qRT-PCR. Additionally, qRT-PCR and western blotting were used to measure STOML2, E-cadherin, and N-cadherin expression in HASMCs. Extracellular matrix components were detected by western blot assay. The viability and migration of HASMCs were analyzed using MTT and Transwell assays. Tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6 contents were evaluated using the corresponding kits. The key molecules of the p38 mitogen-activated protein kinase (p38 MAPK) pathway (p38 and p38) were determined using western blotting. Results Increased STOML2 expression was observed in both patients with asthma and PDGF-BB-treated HASMCs. STOML2 knockdown inhibits STOML2 expression in HASMCs. PDGF-BB induced proliferation, migration, extracellular matrix deposition, inflammatory responses, and p38 MAPK pathway activation in HASMCs. However, the opposite effects were observed following STOML2 knockdown or SB-203580 treatment, respectively. Furthermore, P79350 treatment reversed the effect of STOML2 knockdown. Conclusions Our results showed that silencing STOML2 inhibits inflammation and airway remodeling in PDGF-BB-stimulated HASMCs via the p38 MAPK pathway. Thus, STOML2 is a promising therapeutic target for the treatment of asthma.

To study the expression of stomatin like protein-2 (SLP-2) at mRNA and protein levels in two kinds of malignant epithelial tumors, including laryngeal squamous cell carcinoma (LSCC) and invasive breast cancer, and to study the relations of SLP-2 expression and clinicopathologic parameters with the prognosis. RT-PCR and Western blot were used to detect the expression of SLP-2 mRNA and protein in LSCC and their normal counterparts (46 and 10 pair, respectively). Immunohistochemistry was carried on tissue array constructed from LSCC (104 cases) and breast cancer (263 cases), respectively. The association between SLP-2 expression and clinicopathologic parameters was analyzed. LSCC showed a higher expression of SLP-2 than that of their normal counterparts (negative expression) at mRNA (83%, 38/46) and protein (7/10) level. Immunohistochemical analysis of LSCC showed that compared with negative expression in normal laryngeal epithelium (0/20), a higher SLP-2 expression was detected in LSCC (36/104, P=0.000) and associated with the advanced clinical stage (P<0.01) and lymph node metastasis (P=0.003). Immunohistochemical study of invasive breast cancer demonstrated that compared with negative expression in normal breast tissue (0/10), more than one half of the cases showed a high SLP-2 expression (52.5%, 138/263, P=0.000) in breast cancer, which correlated with the tumor size (P=0.020), lymph node metastasis (P<0.01), advanced clinical stage (P<0.01), distant metastasis (P=0.002) and HER2/neu protein expression (P=0.037). Survival analysis showed a shorter overall survival probability in patients with a high SLP-2 expression. It was considered that lymph node metastasis, positive HER2/neu expression, and high-level SLP-2 expression may act as the independent prognostic factors for those tumors. A high expression level of SLP-2 may be associating with the development of invasion and metastasis in LSCC and breast cancer, and SLP-2 is also considered working as an independent factor indicating a poor prognosis clinically in breast cancer.

Papillary thyroid cancer (PTC) accounts for 80-90% of all cases of thyroid malignancies. Stomatin-like protein 2 (SLP-2) is a novel member of the stomatin superfamily and is found in several types of human tumors. However, whether it is expressed in human PTC is unknown. In the present study, we aimed to explore the diagnostic value of SLP-2 in patients with PTC and to investigate whether SLP-2 expression is regulated by transforming growth factor-β (TGF-β), a cytokine which plays an important role in PTC tumorigenesis. A total of 107 patients consisting of 99 cases of classical and 8 cases of follicular variant PTC was examined. The expression of SLP-2 mRNA and protein was examined by immunohistochemistry (IHC) and qPCR, respectively. We found that SLP-2 was overexpressed in human PTC. The expression of SLP-2 was significantly associated with clinicopathological features of the PTC cases. Particularly, increased SLP-2 expression was mainly correlated with primary tumors >1 cm in size, with late stage tumors and with metastatic lymph nodes. The expression of SLP-2 was correlated with the expression of Ki-67, a cell proliferation marker, in PTC tissues as detected by IHC. SLP-2 was upregulated by TGF-β1 in PTC cells as evaluated by western blotting. The present data revealed for the first time that patients with PTC exhibited SLP-2 overexpression that was associated with clinicopathological features. The correlation between SLP-2 expression and proliferation marker Ki-67 may be characteristic of PTC and may reflect PTC progression. SLP-2 was upregulated by TGF-β1, indicating a possible role of SLP-2 in PTC tumorigenesis. Our data suggest that SLP-2 may be considered as a useful diagnostic marker and therapeutic target for PTC.

Stomatin-like protein 2 (SLP-2) is a member of the highly conserved stomatin protein family whose homologues span from Archaea to humans and include stomatin, SLP-1, and SLP-3. Several studies have indicated that overexpression of SLP-2 is strongly associated with adhesion and migration in several human cancers. The aim of the present study was to evaluate SLP-2 expression at the mRNA and protein level in patients with gastric cancer (GC) and to examine the relationships between SLP-2 expression, clinicopathological features, and prognosis. We investigated SLP-2 expression in primary GC and paired normal gastric tissue by real-time PCR (RT-PCR; n = 16) and Western blot analysis (n = 32). Additionally, we performed immunohistochemistry (IHC) on 113 paraffin-embedded GC specimens, 30 matched normal specimens, and 30 paired metastatic lymph node samples. SLP-2 is overexpressed in GC compared with the adjacent normal gastric epithelium (p < 0.001), and high-level SLP-2 expression is significantly correlated with the depth of invasion, lymph node metastasis, distant metastasis, and American Joint Committee on Cancer (AJCC) stage. Furthermore, elevated SLP-2 expression is an independent prognostic factor in multivariate analysis using the Cox regression model (p = 0.005). Overexpression of SLP-2 may contribute to the progression and poor prognosis of GC.

Several studies have indicated that overexpression of stomatin-like protein 2 (SLP-2) has been identified in several types of cancer. However, its role and clinical relevance in gallbladder cancer (GBC) is unknown. The purpose of this study was to reveal the prognostic significance of SLP-2 in GBC. The SLP-2 expression was examined at mRNA and protein levels by real-time quantitative polymerase chain reaction (qRT-PCR), and immunohistochemistry in GBC tissues and adjacent noncancerous tissues. Statistical analyses were applied to test the associations between SLP-2 expression, clinicopathologic factors, and prognosis. Immunohistochemistry and qRT-PCR showed that the protein and mRNA expression levels of SLP-2 were both significantly higher in GBC tissues than in adjacent noncancerous tissues. In addition, immunohistochemistry analysis showed that SLP-2 expression was significantly correlated with histological grade (P <0.001), pathologic T stage (P = 0.019), clinical stage (P = 0.001), and lymph node metastasis (P = 0.026). The Kaplan-Meier survival curves indicated that patients with high expression of SLP-2 had shorter overall survival than those with low expression (P <0.001). Meanwhile, the Cox multivariate analysis indicated that high expressions of SLP-2 were an independent prognostic factor for patients with GBC. These data showed that SLP-2 may play an important role in human GBC tumorigenesis, and SLP-2 might serve as a novel prognostic marker in human GBC.

Tyrosine kinase inhibitor resistance is a key factor affecting the prognosis of patients with advanced hepatocellular carcinoma (HCC). Previously, our group demonstrated that HCC patients with high stomatin-like protein 2 (STOML2) expression were poorly sensitive to systemic therapy. Whether STOML2 is involved in sorafenib resistance is unclear. Recent mechanistic studies have demonstrated that selective activation of ferroptosis pathways is expected to restore sorafenib sensitivity. The aim of the present study was to investigate the STOML2-ferroptosis axis and its contribution to sorafenib resistance. In this study, STOML2 expression was detected in tissue microarrays from patients with primary HCC and in human cell lines. Functional proliferative clone formation assay was used to study the biological function of STOML2. Ferroptosis was detected by flow cytometry, cellular lipid peroxidation and the malondialdehyde (MDA) test. Western blotting and qPCR assays were used to verify the STOML2-AKT-solute carrier family 7 membrane 11 (SLC7A11) axis and to explore the possible mechanism of the combination of LY294002 (an AKT inhibitor) in patients with advanced HCC. The results indicated that patients with poor efficacy demonstrated higher expression of STOML2 compared with that in samples derived from patients with good efficacy. Knockdown of STOML2 expression inhibited colony formation and IC50 in HCC cell lines treated with sorafenib. High STOML2 expression was negatively correlated with ferroptosis as shown by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. STOML2 inhibited ferroptosis by activating the AKT-SLC7A11 axis, which promoted increased intracellular antioxidant capacity. The AKT inhibitor LY294002 exhibited synergistic antitumor effects with sorafenib in HCC. In conclusion, the present study demonstrated that STOML2 could enhance the AKT-SLC7A11-mediated antioxidant capacity in HCC, inhibit ferroptosis and reduce the sensitivity of HCC to sorafenib, providing a theoretical basis for the combination of LY294002 and sorafenib.

Stomatin-like protein 2 (SLP-2) is overexpressed in numerous types of human cancer and previous studies revealed that SLP-2 may function in mitochondria. The purpose of the present study was to evaluate the expression of SLP-2 in cervical cancer and the association between SLP-2 expression and clinical features, in addition to investigating the role of SLP-2 in the apoptosis of cervical cancer cells. The expression profile of SLP-2 was determined by quantitative polymerase chain reaction, western blotting and immunohistochemical staining. The effect of SLP-2 on cell apoptosis induced by chemotherapeutics in cervical cancer cells was evaluated using Annexin V staining and terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assays. The results indicated that SLP-2 expression in cervical cancer was significantly upregulated at the mRNA and protein levels, compared with that in normal cervical tissues. Immunohistochemical analysis revealed significant correlation between SLP-2 protein expression and clinical characteristics, including the squamous cell carcinoma antigen (P=0.003), deep stromal invasion (P=0.021), lymphovascular space involvement (P=0.044) and pelvic lymph node metastasis (P<0.001), which served as independent prognostic factors for predicting the shortening of overall survival time in patients with early-stage cervical cancer. In addition, TUNEL and Annexin V binding assays revealed that silencing SLP-2 expression significantly enhanced the sensitivity of cervical cancer cells to apoptosis induced by chemotherapeutics. Taken together, the results of the present study suggest that SLP-2 may be a progressive gene in the development of cervical cancer. Overexpression of SLP-2 serves an important role in the apoptosis of human cervical cancer cells.

SLP-2 regulates the generation of reactive oxygen species and the ERK pathway to promote papillary thyroid carcinoma motility and angiogenesis

BackgroundLoss-of-function mutations in the PRKN gene, encoding Parkin, are the most common cause of autosomal recessive Parkinson’s disease (PD). We have previously identified mitochondrial Stomatin-like protein 2 (SLP-2), which functions in the assembly of respiratory chain proteins, as a Parkin-binding protein. Selective knockdown of either Parkin or SLP-2 led to reduced mitochondrial and neuronal function in neuronal cells and Drosophila, where a double knockdown led to a further worsening of Parkin-deficiency phenotypes. Here, we investigated the minimal Parkin region involved in the Parkin-SLP-2 interaction and explored the ability of Parkin-fragments and peptides from this minimal region to restore mitochondrial function.MethodsIn fibroblasts, human induced pluripotent stem cell (hiPSC)-derived neurons, and neuroblastoma cells the interaction between Parkin and SLP-2 was investigated, and the Parkin domain responsible for the binding to SLP-2 was mapped. High resolution respirometry, immunofluorescence analysis and live imaging were used to analyze mitochondrial function.ResultsUsing a proximity ligation assay, we quantitatively assessed the Parkin-SLP-2 interaction in skin fibroblasts and hiPSC-derived neurons. When PD-associated PRKN mutations were present, we detected a significantly reduced interaction between the two proteins. We found a preferential binding of SLP-2 to the N-terminal part of Parkin, with a highest affinity for the RING0 domain. Computational modeling based on the crystal structure of Parkin protein predicted several potential binding sites for SLP-2 within the Parkin RING0 domain. Amongst these, three binding sites were observed to overlap with natural PD-causing missense mutations, which we demonstrated interfere substantially with the binding of Parkin to SLP-2. Finally, delivery of the isolated Parkin RING0 domain and a Parkin mini-peptide, conjugated to cell-permeant and mitochondrial transporters, rescued compromised mitochondrial function in Parkin-deficient neuroblastoma cells and hiPSC-derived neurons with endogenous, disease causing PRKN mutations.ConclusionsThese findings place further emphasis on the importance of the protein–protein interaction between Parkin and SLP-2 for the maintenance of optimal mitochondrial function. The possibility of restoring an abolished binding to SLP-2 by delivering the Parkin RING0 domain or the Parkin mini-peptide involved in this specific protein–protein interaction into cells might represent a novel organelle-specific therapeutic approach for correcting mitochondrial dysfunction in Parkin-linked PD.

Stomatin-like protein 2 (SLP-2) is a member of the stomatin – prohibitin – flotillin – HflC/K (SPFH) superfamily. Recent evidence indicates that SLP-2 is involved in the organization of cardiolipin-enriched microdomains in mitochondrial membranes and the regulation of mitochondrial biogenesis and function. In T cells, this role translates into enhanced T cell activation. Although the major pool of SLP-2 is associated with mitochondria, we show here that there is an additional pool of SLP-2 associated with the plasma membrane of T cells. Both plasma membrane-associated and mitochondria-associated pools of SLP-2 coalesce at the immunological synapse (IS) upon T cell activation. SLP-2 is not required for formation of IS nor for the re-localization of mitochondria to the IS because SLP-2-deficient T cells showed normal re-localization of these organelles in response to T cell activation. Interestingly, upon T cell activation, we found the surface pool of SLP-2 mostly excluded from the central supramolecular activation complex, and enriched in the peripheral area of the IS where signalling TCR microclusters are located. Based on these results, we propose that SLP-2 facilitates the compartmentalization not only of mitochondrial membranes but also of the plasma membrane into functional microdomains. In this latter location, SLP-2 may facilitate the optimal assembly of TCR signalosome components. Our data also suggest that there may be a net exchange of membrane material between mitochondria and plasma membrane, explaining the presence of some mitochondrial proteins in the plasma membrane.

Enhanced SLP-2 promotes invasion and metastasis by regulating Wnt/β-catenin signal pathway in colorectal cancer and predicts poor prognosis

To explore the expression of Stomatin-like protein 2 (SLP-2) and its clinical significance in epithelial ovarian cancer (EOC). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the differential expression of SLP-2 in EOC tissues and cell lines. The relationship between SLP-2 expression and clinical pathological data of EOC patients was analyzed. QRT-PCR results suggested that the SLP-2 was up-regulated in both EOC tissues and EOC cells by comparing with normal control. SLP-2 expression was a correlation with tumor pathological grade, distant metastasis, and TNM stage in EOC patients. Down-regulation of SLP-2 could significantly inhibit proliferation and promote apoptosis of EOC cells by activating the Notch signaling pathway. Knockdown of SLP-2 markedly downregulated Notch1 and Hes1. SLP-2 was a novel factor involved in EOC progression, and could be utilized as a potential biomarker and therapeutic target for the EOC patients.

We have previously reported that stomatin-like protein 2 (SLP-2) regulates human T cell activation through the antigen receptor. In human lymphoid tissues, SLP-2 is widely expressed in the thymus, lymph nodes and tonsils, and its expression is further up-regulated in response to lymphocyte activation. In T cells, SLP-2 is found in two pools: the most abundant is associated with mitochondria while a smaller pool is associated with the plasma membrane. To further investigate the role of SLP-2, we generated T cell-specific SLP-2 deficient mice. These mice had normal numbers of thymocytes and mature T cells. However, SLP-2 deficient T cells showed significantly less T cell activation in response to T cell stimulation in vivo and in vitro. Although SLP-2 deficient T cells did not show significant alterations in mitochondrial biogenesis, they had decreased levels of the NDUSF3, NDUFB8 and NDUFA9 subunits of complex I of the respiratory chain, suggesting a possible role for SLP-2 in regulating the stability of proteins involved in mitochondrial electron transport. SLP-2 deficient T cells also had less cardiolipin within detergent-insoluble microdomains, which may be required for proper respiratory chain assembly and function. Based on these results, we conclude that SLP-2 contributes to T cell activation, in part by regulating stability of complex I of the respiratory chain and sustaining the metabolic requirements for T cell activation.