Lysosomal cyst(e)ine storage potentiates tolerance to oxidative stress in cancer cells.

Cyst(e)ine is a key precursor for the synthesis of glutathione (GSH), which protects cancer cells from oxidative stress. Cyst(e)ine is stored in lysosomes, but its role in redox regulation is unclear. Here, we show that breast cancer cells upregulate major facilitator superfamily domain containing 12 (MFSD12) to increase lysosomal cyst(e)ine storage, which is released by cystinosin (CTNS) to maintain GSH levels and buffer oxidative stress. We find that mTORC1 regulates MFSD12 by directly phosphorylating residue T254, while mTORC1 inhibition enhances lysosome acidification that activates CTNS. This switch modulates lysosomal cyst(e)ine levels in response to oxidative stress, fine-tuning redox homeostasis to enhance cell fitness. MFSD12-T254A mutant inhibits MFSD12 function and suppresses tumor progression. Moreover, MFSD12 overexpression correlates with poor neoadjuvant chemotherapy response and prognosis in breast cancer patients. Our findings reveal the critical role of lysosomal cyst(e)ine storage in adaptive redox homeostasis and suggest that MFSD12 is a potential therapeutic target.

The phospholipid flippase ATP9A enhances macropinocytosis to promote nutrient starvation tolerance in hepatocellular carcinoma.

Macropinocytosis is an effective strategy to mitigate nutrient starvation. It can fuel cancer cell growth in nutrient-limited conditions. However, whether and how macropinocytosis contributes to the rapid proliferation of hepatocellular carcinoma cells, which frequently experience an inadequate nutrient supply, remains unclear. Here, we demonstrated that nutrient starvation strongly induced macropinocytosis in some hepatocellular carcinoma cells. It allowed the cells to acquire extracellular nutrients and supported their energy supply to maintain rapid proliferation. Furthermore, we found that the phospholipid flippase ATP9A was critical for regulating macropinocytosis in hepatocellular carcinoma cells and that high ATP9A levels predicted a poor outcome for patients with hepatocellular carcinoma. ATP9A interacted with ATP6V1A and facilitated its transport to the plasma membrane, which promoted plasma membrane cholesterol accumulation and drove RAC1-dependent macropinocytosis. Macropinocytosis inhibitors significantly suppressed the energy supply and proliferation of hepatocellular carcinoma cells characterised by high ATP9A expression under nutrient-limited conditions. These results have revealed a novel mechanism that overcomes nutrient starvation in hepatocellular carcinoma cells and have identified the key regulator of macropinocytosis in hepatocellular carcinoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Modifiable risk factors mediate the effect of gastroesophageal reflux disease on stroke and subtypes: A Mendelian randomization study.

OBJECTIVES: Previous observational studies have suggested that gastroesophageal reflux disease (GERD) increases the risk of stroke, but the specific underlying mechanisms are unclear. We investigated the causal associations of GERD with stroke and its subtypes using Mendelian randomization (MR), and evaluated the potential mediating effects of modifiable stroke risk factors in the causal pathway. METHODS: Genetic instrumental variables for GERD were extracted from the latest genome-wide association study (GWAS) summary level data. We initially performed two-sample MR to examine the association of GERD with stroke and its subtypes, including ischemic stroke, intracranial hemorrhage, and the major subtypes of ischemic stroke. Two-step MR was further employed to investigate the mediating effect of 15 risk factors in the causal pathway. RESULTS: We found significant causal associations of genetically predicted GERD with increased risk of stroke (OR: 1.22 95% CI: 1.126-1.322), ischemic stroke (OR: 1.19 95% CI: 1.098-1.299), and large-artery stroke (OR: 1.49 95% CI: 1.214-1.836). Replication and sensitivity analyses yielded consistent effect directions and similar estimates. Further mediation analyses indicated that hypertension (HTN), systolic blood pressure (SBP), and type 2 diabetes (T2D) mediated 36.0%, 9.0%, and 15.8% of the effect of GERD on stroke; 42.9%, 10.8%, and 21.4% for ischemic stroke, and 23.3%; 7.9%, and 18.7% for large-artery stroke, respectively. CONCLUSIONS: This study supports that GERD increases susceptibility to stroke, ischemic stroke, and large-artery stroke, and is partially mediated by HTN, SBP, and T2D.

Minimum taxi fleet algorithm considering human spatiotemporal behaviors.

With the development of information technology, more and more travel data have provided great convenience for scholars to study the travel behavior of users. Planning user travel has increasingly attracted researchers' attention due to its great theoretical significance and practical value. In this study, we not only consider the minimum fleet size required to meet the urban travel needs but also consider the travel time and distance of the fleet. Based on the above reasons, we propose a travel scheduling solution that comprehensively considers time and space costs, namely, the Spatial-Temporal Hopcroft-Karp (STHK) algorithm. The analysis results show that the STHK algorithm not only significantly reduces the off-load time and off-load distance of the fleet travel by as much as 81% and 58% and retains the heterogeneous characteristics of human travel behavior. Our study indicates that the new planning algorithm provides the size of the fleet to meet the needs of urban travel and reduces the extra travel time and distance, thereby reducing energy consumption and reducing carbon dioxide emissions. Concurrently, the travel planning results also conform to the basic characteristics of human travel and have important theoretical significance and practical application value.

Rhophilin-associated tail protein 1 promotes migration and metastasis in triple negative breast cancer via activation of RhoA.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with high motile and invasive capacity that contributes to metastasis. Understanding the mechanisms for the motility of TNBC might provide novel targetable vulnerabilities of the tumors. Herein, we find that Rhophilin-associated tail protein 1 (ROPN1) is selectively overexpressed in human TNBC cell lines and tissues. Overexpression of ROPN1 promotes, while silencing of ROPN1 inhibits the robust migration, invasion, and in vivo metastasis of TNBC cells. Moreover, we find that ROPN1 activates RhoA signaling via rhophilin-1 (RHPN1), leading to enhanced actin stress fibers formation in TNBC cells. RhoA signaling is demonstrated to be essential for ROPN1-mediated migration and metastasis of TNBC cells. Finally, we find that high levels of ROPN1 are significantly associated distant metastasis and predicted poor prognosis in patients with breast cancer. These findings reveal a novel mechanism for the high motility and metastasis of TNBC cells, suggesting that ROPN1 might be a potential prognostic marker and therapeutic target.

HN1 promotes tumor associated lymphangiogenesis and lymph node metastasis via NF-κB signaling activation in cervical carcinoma.

Lymph node metastasis (LNM) is a critical cause for disease progression and treatment failure in cervical cancer. However, the mechanism underlying cervical cancer LNM remains unclear. In this study, HN1 was found to be dramatically upregulated in cervical cancer and patients with higher HN1 expression are more likely to exhibit a higher rate of LNM and lower survival rate. Univariate and multivariate Cox-regression analyses showed that HN1 is an independent prognostic factor in cervical cancer. Meanwhile, HN1 promotes lymphangiogenesis of cervical cancer in vitro. The in vivo experiment also indicates that HN1 enhances LNM in cervical cancer. Furthermore, we also found that HN1 activated the NF-κB signaling pathway to enhance the expression of downstream genes. Taken together, our study suggests that HN1 plays a crucial role in promoting LNM and acts as a prognostic biomarker in cervical cancer.

Nuclear orphan receptor NR2F6 confers cisplatin resistance in epithelial ovarian cancer cells by activating the Notch3 signaling pathway.

The primary challenge facing treatment of epithelial ovarian cancer (EOC) is the high frequency of chemoresistance, which severely impairs the quality of life and survival of patients with EOC. Our study aims to investigate the mechanisms by which upregulation of NR2F6 induces chemoresistance in EOC. The biological roles of NR2F6 in EOC chemoresistance were explored in vitro by Sphere, MTT and AnnexinV/PI assay, and in vivo using an ovarian cancer orthotopic transplantation model. Bioinformatics analysis, luciferase assay, CHIP and IP assays were performed to identify the mechanisms by which NR2F6 promotes chemoresistance in EOC. The expression of NR2F6 was significantly upregulated in chemoresistant EOC tissue, and NR2F6 expression was correlated with poorer overall survival. Moreover, overexpression of NR2F6 promotes the EOC cancer stem cell phenotype; conversely, knockdown of NR2F6 represses the EOC cancer stem cell phenotype and sensitizes EOC to cisplatin in vitro and in vivo. Our results further demonstrate that NR2F6 sustains activated Notch3 signaling, resulting in chemoresistance in EOC cells. Notably, NR2F6 acts as an informative biomarker to identify the population of EOC patients who are likely to experience a favorable objective response to gamma-secretase inhibitors (GSI), which inhibit Notch signaling. Therefore, concurrent inhibition of NR2F6 and treatment with GSI and cisplatin-based chemotherapy may be a novel therapeutic approach for NR2F6-overexpressing EOC. In summary, we have, for the first time, identified an important role for NR2F6 in EOC cisplatin resistance. Our study suggests that GSI may serve as a potential targeted treatment for patients with NR2F6-overexpressing EOC.

Synaptopodin-2 suppresses metastasis of triple-negative breast cancer via inhibition of YAP/TAZ activity.

Triple-negative breast cancer (TNBC) represents the most aggressive subtype of breast cancer, with a high incidence of distant metastasis; however, the underlying mechanism for this frequent recurrence remains unclear. Herein, we show that synaptopodin-2 (SYNPO2), a putative tumour suppressor in aggressive cancer, is frequently downregulated in TNBC by methylation of the promoter of SYNPO2. Low expression levels of SYNPO2 correlated significantly with 5-year metastatic relapse, and predicted poorer prognosis in breast cancer patients. Reintroduction of SYNPO2 inhibited the invasion and spontaneous metastasis of TNBC cells in vivo. Strikingly, downregulation of SYNPO2 is essential for the maintenance of stem cell-like properties in TNBC cells, leading to efficient distant colonization and metastasis outgrowth. Moreover, we demonstrate that SYNPO2 inhibits the activities of YAP and TAZ by stabilizing LATS2 protein, and transduction of YAP-S127A abrogates the repressive role of SYNPO2 in metastasis. Finally, immunohistochemical (IHC) analysis of breast cancer patient specimens indicated that the SYNPO2-LATS2-YAP axis is clinically relevant. These findings uncover a suppressive role of SYNPO2 in TNBC metastasis via inhibition of YAP/TAZ, and suggest that SYNPO2 might provide a potential prognosis marker and novel therapeutic strategy. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Overexpression of CDCA7 predicts poor prognosis and induces EZH2-mediated progression of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with high proliferative and metastatic phenotypes. CDCA7, a new member of the cell division cycle associated family of genes, is involved in embryonic development and dysregulated in various types of human cancer. However, the biological role and molecular mechanism of CDCA7 in TNBC have not been defined. Herein, we found that CDCA7 was preferentially and markedly expressed in TNBC cell lines and tissues. High expression of CDCA7 was associated with metastatic relapse status and predicted poorer disease-free survival in patients with TNBC. We observed that CDCA7 silencing in TNBC cell lines effectively impaired cell proliferation, invasion and migration in vitro. Importantly, depletion of CDCA7 strongly reduced the tumorigenicity and distant colonization capacities of TNBC cells in vivo. Furthermore, CDCA7 increased the expression of EZH2, a marker of aggressive breast cancer that is involved in tumor progression, by enhancing the transcriptional activity of its promoter. This increase in EZH2 expression was essential for the CDCA7-mediated effects on TNBC progression. Finally, our immunohistochemical analysis revealed that the CDCA7/EZH2 axis was clinical relevant. These findings suggest CDCA7 plays a crucial role in TNBC progression by transcriptionally upregulating EZH2 and might be a potential prognostic factor and therapeutic target in TNBC.

Thymosin beta 10 is a key regulator of tumorigenesis and metastasis and a novel serum marker in breast cancer.

BACKGROUND: Thymosin beta 10 (TMSB10) has been demonstrated to be involved in the malignant process of many cancers. The purpose of this study was to determine the biological roles and clinical significance of TMSB10 in breast cancer and to identify whether TMSB10 might be used as a serum marker for the diagnosis of breast cancer. METHODS: TMSB10 expression was evaluated by immunohistochemical analysis (IHC) of 253 breast tumors and ELISA of serum from 80 patients with breast cancer. Statistical analysis was performed to explore the correlation between TMSB10 expression and clinicopathological features in breast cancer. Univariate and multivariate Cox regression analysis were performed to examine the association between TMSB10 expression and overall survival and metastatic status. In vitro and in vivo assays were performed to assess the biological roles of TMSB10 in breast cancer. Western blotting and luciferase assays were examined to identify the underlying pathway involved in the tumor-promoting role of TMSB10. RESULTS: We found TMSB10 was upregulated in breast cancer cells and tissues. Univariate and multivariate analysis demonstrated that high TMSB10 expression significantly correlated with clinicopathological features, poor prognosis and distant metastases in patients with breast cancer. Overexpression of TMSB10 promotes, while silencing of TMSB10 inhibits, proliferation, invasion and migration of breast cancer cells in vitro and in vivo. Our results further reveal that TMSB10 promotes the proliferation, invasion and migration of breast cancer cells via AKT/FOXO signaling, which is antagonized by the AKT kinase inhibitor perifosine. Importantly, the expression of TMSB10 is significantly elevated in the serum of patients with breast cancer and is positively associated with clinical stages of breast cancer. CONCLUSION: TMSB10 may hold promise as a minimally invasive serum cancer biomarker for the diagnosis of breast cancer and a potential therapeutic target which will facilitate the development of a novel therapeutic strategy against breast cancer.

The TGF-ß signalling negative regulator PICK1 represses prostate cancer metastasis to bone.

Backgroud:Constitutive activation of TGF-ß signalling is a well-recognised mechanism in bone metastasis of prostate cancer (PCa). Protein Interacting with PRKCA 1 (PICK1) is a critical negative regulator of the TGF-ß pathway. However, the clinical significance and biological role of PICK1 in PCa bone metastasis remain obscure. METHODS: PICK1 expression is evaluated by immunohistochemistry (IHC) in 198 PCa patients. Statistical analysis is performed to explore correlation between PICK1 expression and clinicopathological features in PCa patients. The biological role of PICK1 is examined in PC-3 and C4-2B cells in vitro and a mouse intracardial model in vivo. RESULTS: PICK1 expression is decreased in PCa tissues with bone metastasis and bone-derived cells and downregulation of PICK1 positively correlates with serum PSA level, Gleason grade and bone metastasis status in PCa patients. Overexpression of PICK1 suppresses PCa cell invasion and migration in vitro and bone metastasis in vivo. Our results further indicate downregulation of PICK1 is caused by miR-210-3p overexpression in PCa tissues with bone metastasis. Clinical negative correlation of PICK1 with miR-210-3p is confirmed in PCa tissues. CONCLUSIONS: Our findings uncover a novel functionally and clinically relevant epigenetic regulatory mechanism for constitutive activation of TGF-ß signalling in bone metastasis of PCa.

Prostate tumour overexpressed-1 promotes tumourigenicity in human breast cancer via activation of Wnt/ß-catenin signalling.

Breast cancer is the most common malignancy in females. The presence of cancer stem cells (CSCs) is the main cause of local and distant tumour recurrence and is associated with poor outcome in breast cancer. However, the molecular mechanisms underlying the maintenance of CSCs remain largely unknown. This study demonstrates that prostate tumour overexpressed-1 (PTOV1) enhances the CSC population and augments the tumourigenicity of breast cancer cells both in vitro and in vivo. Moreover, PTOV1 suppresses transcription of Dickkopf-1 (DKK1) by recruiting histone deacetylases and subsequently reducing DKK1 promoter histone acetylation, followed by activation of Wnt/ß-catenin signalling. Restoration of DKK1 expression in PTOV1-overexpressing cells counteracts the effects of PTOV1 on Wnt/ß-catenin activation and the CSC population. Collectively, these results suggest that PTOV1 positively regulates the Wnt/ß-catenin signalling pathway and enhances tumourigenicity in breast cancer; this novel mechanism may represent a therapeutic target for breast cancer. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

TBL1XR1 promotes lymphangiogenesis and lymphatic metastasis in esophageal squamous cell carcinoma.

OBJECTIVE: Transducin (ß)-like 1 X-linked receptor 1 (TBL1XR1) plays an important role in controlling the precisely regulated switch between gene repression and gene activation in transcriptional regulation. We investigated its biological function and clinical significance in esophageal squamous cell carcinoma (ESCC). DESIGN: Immunoblotting and immunochemistry were used to determine TBL1XR1 expression in ESCC cell lines, ESCC clinical tissues and 230 clinicopathologically characterised ESCC specimens. The role of TBL1XR1 in lymphangiogenesis and lymphatic metastasis was examined by tube formation, cell invasion and wound-healing assays in vitro, and by a popliteal lymph node metastasis model in vivo. The molecular mechanism by which TBL1XR1 upregulates vascular endothelial growth factor C (VEGF-C) expression was explored using real-time PCR, ELISA, luciferase reporter assay and chromatin immunoprecipitation. RESULTS: TBL1XR1 expression was significantly upregulated in ESCC, positively correlated with disease stage and patient survival, and identified as an independent prognostic factor for patient outcome. We found that TBL1XR1 overexpression promoted lymphangiogenesis and lymphatic metastasis in ESCC in vitro and in vivo, whereas TBL1XR1 silencing had the converse effect. We demonstrated that TBL1XR1 induced VEGF-C expression by binding to the VEGF-C promoter. We confirmed the correlation between TBL1XR1 and VEGF-C expression in a large cohort of clinical ESCC samples and through analysis of published datasets in gastric, colorectal and breast cancer. CONCLUSIONS: Our results demonstrated that TBL1XR1 induced lymphangiogenesis and lymphatic metastasis in ESCC via upregulation of VEGF-C, and may represent a novel prognostic biomarker and therapeutic target for patients with ESCC.

Transducin (ß)-like 1 X-linked receptor 1 promotes proliferation and tumorigenicity in human breast cancer via activation of beta-catenin signaling.

INTRODUCTION: Transducin (ß)-like 1 X-linked receptor 1(TBLR1) is an F-box-like and WD repeat-containing protein which functions as a switch in transcriptional activation, However, the clinical significance and biological role of TBLR1 in breast cancer remains largely unknown. METHODS: Western blotting, immunocytochemistry and real-time PCR were used to evaluate TBLR1 expression in normal breast epithelial cells and breast cancer cell lines, clinical tissue samples and adjacent nontumor tissues, and in 214 paraffin-embedded specimens. Statistical analyses were used to test for the prognostic and diagnostic associations. The biological role of TBLR1 -induced proliferation and tumorigenicity in breast cancer cells was explored in vitro and in vivo. The effect of TBLR1 on the expression of cyclin D1 and ß-catenin signaling was examined by Western blotting, luciferase reporter assay and by several immunoprecipitation techniques. RESULTS: TBLR1 was significantly upregulated in breast cancer cells and tissues compared to normal control samples. Immunohistochemical analysis revealed high expression of TBLR1 in 113 of 214 (52.8%) paraffin-embedded archival breast cancer. The overall expression level of TBLR1 was significantly correlated with clinical stage (P <0.001), the tumor classification (P <0.001), node classification (P =0.024), and metastasis classification (P = 0.004), histological grade (P = 0.044), as well as with the expression level of c-erbB2 (P = 0.036) and Ki-67 (P <0.001). Patients with higher TBLR1 expression had shorter overall survival time, whereas patients with lower TBLR1 expression had better survival. Multivariate analysis suggested that TBLR1 expression might be an independent prognostic indicator for the survival of breast cancer patients. TBLR1 overexpression promoted, whereas TBLR1 silencing inhibited, proliferation and tumorigenicity in breast cancer cells both in vitro and in vivo. We found that TBLR1 expression was implicated in the upregulation of cyclin D1, phosphorylation of cell-cycle control protein Rb (pRb) and activation of ß-catenin signaling in breast cancer. CONCLUSIONS: TBLR1 plays a key role in the development and progression of breast cancer cells via cyclin D1-transactivation and activation of the ß-catenin signaling pathway. TBLR1 may be a novel prognostic marker and a potential therapeutic target in the treatment human breast cancer.

Acylglycerol kinase promotes cell proliferation and tumorigenicity in breast cancer via suppression of the FOXO1 transcription factor.

BACKGROUND: Acylglycerol kinase (AGK) is reported to be overexpressed in multiple cancers. The clinical significance and biological role of AGK in breast cancer, however, remain to be established. METHODS: AGK expression in breast cancer cell lines, paired patient tissues were determined using immunoblotting and Real-time PCR. 203 human breast cancer tissue samples were analyzed by immunochemistry (IHC) to investigate the relationship between AGK expression and the clinicopathological features of breast cancer. Functional assays, such as colony formation, anchorage-independent growth and BrdU assay, and a xenograft tumor model were used to determine the oncogenic role of AGK in human breast cancer progression. The effect of AGK on FOXO1 transactivity was further investigated using the luciferase reporter assays, and by detection of the FOXO1 downstream genes. RESULTS: Herein, we report that AGK was markedly overexpressed in breast cancer cells and clinical tissues. Immunohistochemical analysis showed that the expression of AGK significantly correlated with patients' clinicopathologic characteristics, including clinical stage and tumor-nodule-metastasis (TNM) classification. Breast cancer patients with higher levels of AGK expression had shorter overall survival compared to patients with lower AGK levels. We gained valuable insights into the mechanism of AGK expression in breast cancer cells by demonstrating that overexpressing AGK significantly enhanced, whereas silencing endogenous AGK inhibited, the proliferation and tumorigenicity of breast cancer cells both in vitro and in vivo. Furthermore, overexpression of AGK enhanced G1-S phase transition in breast cancer cells, which was associated with activation of AKT, suppression of FOXO1 transactivity, downregulation of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1 and upregulation of the cell cycle regulator cyclin D1. CONCLUSIONS: Taken together, these findings provide new evidence that AGK plays an important role in promoting proliferation and tumorigenesis in human breast cancer and may serve as a novel prognostic biomarker and therapeutic target in this disease.

Dual effects of serum urate on stroke risk and prognosis: insights from Mendelian randomization.

Background: To investigate the causal associations of serum urate (SUA) with stroke risk and prognosis using Mendelian randomization (MR) and the potential mediating role of stroke risk factors in the causal pathways. Methods: We used the random-effects inverse variance weighting (IVW) as our primary method. We initially performed two-sample univariable MR (UVMR) to identify the causal associations of SUA (n = 437,354) with any stroke (AS, FinnGen: n = 311,635; MEGASTROKE: n = 446,696), ischemic stroke (IS, FinnGen: n = 212,774; MEGASTROKE: n = 440,328), intracranial hemorrhage (ICH, FinnGen: n = 343,663; ISGC: n = 3,026), functional outcome after ischemic stroke at 90d (n = 4,363), and motor recovery within 24 months after stroke (n = 488), and then multivariable MR (MVMR) to estimate the direct causal effects of SUA on these outcomes, adjusting for potential confounders. Finally, we further conducted a two-step MR to investigate the potential mediating role of body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), and estimated glomerular filtration rate (eGFR) in the identified causal pathways. Results: Genetically predicted elevated SUA levels were significantly associated with increased risk of AS (meta-analysis: OR = 1.09, 95% CI [1.04-1.13], p = 3.69e-05) and IS (meta-analysis: OR = 1.10, 95% CI [1.01-1.19], p = 0.021) and with improved poor functional outcome after ischemic stroke at 90d (OR = 0.81, 95% CI [0.72-0.90], p = 1.79e-04) and motor recovery within 24 months after stroke (OR = 1.42, 95% CI [1.23-1.64], p = 2.15e-06). In MVMR, SBP and DBP significantly attenuated the causal effects of SUA on AS, IS, and functional outcome after ischemic stroke at 90d and motor recovery within 24 months after stroke. Further mediation analyses showed that SBP mediated 52.4 and 34.5% of the effects of SUA on AS and IS, while DBP mediated 28.5 and 23.4% of the causal effects, respectively. Conclusion: This study supports the dual role of genetically predicted SUA in increasing stroke risk, especially ischemic stroke risk, and in improving functional outcome and motor recovery. SBP and DBP are key mediators lying on the causal pathways of SUA with AS and IS.

Flotillin-1 promotes tumor necrosis factor-α receptor signaling and activation of NF-κB in esophageal squamous cell carcinoma cells.

BACKGROUND & AIMS: The flotillin family of proteins, including flotillin-1 (FLOT1 or Reggie-2), are lipid raft proteins that initiate receptor kinase signaling and are up-regulated in several tumor types. We investigated the role of FLOT1 signaling and activation of the transcription factor nuclear factor (NF)-κB in esophageal squamous cell carcinoma (ESCC) cells. METHODS: We used immunoblot and immunochemical analyses to determine levels of the lipid raft-associated protein FLOT1 in ESCC cell lines and 432 ESSC samples from patients; primary normal esophageal epithelial cells and matched adjacent nontumor tissues were used as controls. We determined the ability of FLOT1 to activate NF-κB using kinase, electrophoretic mobility shift, and luciferase reporter assays. We measured the effects of FLOT1 overexpression and knockdown with short hairpin RNAs in ESCC cell lines using colony formation, anchorage-independent growth, chicken chorioallantoic membrane, transwell matrix penetration, and Annexin V-binding assays. We analyzed growth of ESCC xenograft tumors in nude mice. RESULTS: Levels of FLOT1 were increased in ESCC cell lines and samples from patients, compared with controls; protein levels correlated with disease stage and survival time. Overexpression of FLOT1 in Kyse30 and Kyse510 ESCC cell lines increased proliferation, anchorage-independent growth, and invasive activity and protected them from apoptosis. FLOT1-transduced ESCC cells formed larger tumors in nude mice than control cells (transduced with only the vector). FLOT1 facilitated recruitment of the tumor necrosis factor-α receptor to lipid rafts; promoted K63-linked polyubiquitination of the signaling intermediaries tumor necrosis factor receptor associated factor 2, receptor interacting protein, and NEMO; and sustained the activation of NF-κB. Levels of FLOT1 correlated with activation of NF-κB in ESCC samples from patients. CONCLUSIONS: The lipid raft protein FLOT1 is up-regulated in ESCC cell lines and samples from patients and promotes ESCC cell proliferation and tumor growth in mice. FLOT1 activates tumor necrosis factor-α receptor signaling and sustains activation of NF-κB in ESCC cells.

Knockdown of stomatin-like protein 2 (STOML2) reduces the invasive ability of glioma cells through inhibition of the NF-κB/MMP-9 pathway.

Stomatin-like protein 2 (STOML2), a member of the stomatin family, has been reported to be up-regulated in several types of human cancers. The clinical significance and biological role of STOML2 in gliomas remain largely unknown. Here, we describe the significantly up-regulated expression of STOML2 in glioma cell lines and glioma tissues at both the transcriptional and translational levels. Silencing endogenous STOML2 in glioma cells and primary glioma cells drastically reduced their migratory speed and invasive ability, associated with induction of matrix metallopeptidase 9 (MMP-9). We also demonstrated that knockdown of STOML2 significantly inhibited the transcriptional activity of NF-κB and repressed the expression levels of NF-κB target genes, including MMP-9. A luciferase reporter assay revealed that the impact of STOML2 on MMP-9 expression is NF-κB-dependent. Immunohistochemical analysis showed that the up-regulation of STOML2 was significantly correlated with the WHO histological grade of gliomas (p < 0.001). Patients with higher STOML2 expression levels had an overall shorter survival time, whereas patients with lower expression of STOML2 had a longer survival time. A multivariate analysis revealed that STOML2 expression might be an independent prognostic indicator for the survival of glioma patients. Taken together, our results suggest that overexpression of STOML2 is associated with glioma aggressiveness and may represent an independent prognostic factor for the outcome of glioma patients.

Inhibition of DPAGT1 suppresses HER2 shedding and trastuzumab resistance in human breast cancer.

Human epidermal growth factor receptor 2-targeted (HER2-targeted) therapy is the mainstay of treatment for HER2+ breast cancer. However, the proteolytic cleavage of HER2, or HER2 shedding, induces the release of the target epitope at the ectodomain (ECD) and the generation of a constitutively active intracellular fragment (p95HER2), impeding the effectiveness of anti-HER2 therapy. Therefore, identifying key regulators in HER2 shedding might provide promising targetable vulnerabilities against resistance. In the current study, we found that upregulation of dolichyl-phosphate N-acetylglucosaminyltransferase (DPAGT1) sustained high-level HER2 shedding to confer trastuzumab resistance, which was associated with poor clinical outcomes. Upon trastuzumab treatment, the membrane-bound DPAGT1 protein was endocytosed via the caveolae pathway and retrogradely transported to the ER, where DPAGT1 induced N-glycosylation of the sheddase - ADAM metallopeptidase domain 10 (ADAM10) - to ensure its expression, maturation, and activation. N-glycosylation of ADAM10 at N267 protected itself from ER-associated protein degradation and was essential for DPAGT1-mediated HER2 shedding and trastuzumab resistance. Importantly, inhibition of DPAGT1 with tunicamycin acted synergistically with trastuzumab treatment to block HER2 signaling and reverse resistance. These findings reveal a prominent mechanism for HER2 shedding and suggest that targeting DPAGT1 might be a promising strategy against trastuzumab-resistant breast cancer.

BAG2 drives chemoresistance of breast cancer by exacerbating mutant p53 aggregate.

Rationale: Chemoresistance is a major challenge in the clinical management of patients with breast cancer. Mutant p53 proteins tend to form aggregates that promote tumorigenesis in cancers. We here aimed to explore the mechanism for the generation of mutant p53 aggregates in breast cancer and assess its role in inducing chemoresistance. Methods: Expression of BCL2-associated athanogene 2 (BAG2) was evaluated by qRT-PCR, western blotting, and immunohistochemistry in breast cancer patient specimens. The significance of BAG2 expression in prognosis was assessed by Kaplan-Meier survival analysis and the Cox regression model. The roles of BAG2 in facilitating the formation of mutant p53 aggregates were analyzed by co-immunoprecipitation, immunofluorescence, and semi-denaturing detergent-agarose gel electrophoresis assays. The effects of BAG2 on the chemoresistance of breast cancer were demonstrated by cell function assays and mice tumor models. Results: In the present study, we found that BAG2 was significantly upregulated in relapse breast cancer patient tissues and high BAG2 was associated with a worse prognosis. BAG2 localized in mutant p53 aggregates and interacted with misfolded p53 mutants. BAG2 exacerbated the formation of the aggregates and recruited HSP90 to promote the propagation and maintenance of the aggregates. Consequently, BAG2-mediated mutant p53 aggregation inhibited the mitochondrial apoptosis pathway, leading to chemoresistance in breast cancer. Importantly, silencing of BAG2 or pharmacological targeting of HSP90 substantially reduced the aggregates and increased the sensitivity of chemotherapy in breast cancer. Conclusion: These findings reveal a significant role of BAG2 in the chemoresistance of breast cancer via exacerbating mutant p53 aggregates and suggest that BAG2 may serve as a potential therapeutic target for breast cancer patients with drug resistance.