SHC4 orchestrates ß-catenin pathway-mediated metastasis in triple-negative breast cancer by promoting Src kinase autophosphorylation.

Triple-negative breast cancer (TNBC) is highly aggressive and metastatic, and has the poorest prognosis among all breast cancer subtypes. Activated ß-catenin is enriched in TNBC and involved in Wnt signaling-independent metastasis. However, the underlying mechanisms of ß-catenin activation in TNBC remain unknown. Here, we found that SHC4 was upregulated in TNBC and high SHC4 expression was significantly correlated with poor outcomes. Overexpression of SHC4 promoted TNBC aggressiveness in vitro and facilitated TNBC metastasis in vivo. Mechanistically, SHC4 interacted with Src and maintained its autophosphorylated activation, which activated ß-catenin independent of Wnt signaling, and finally upregulated the transcription and expression of its downstream genes CD44 and MMP7. Furthermore, we determined that the PxPPxPxxxPxxP sequence on CH2 domain of SHC4 was critical for SHC4-Src binding and Src kinase activation. Overall, our results revealed the mechanism of ß-catenin activation independent of Wnt signaling in TNBC, which was driven by SHC4-induced Src autophosphorylation, suggesting that SHC4 might be a potential prognostic marker and therapeutic target in TNBC.

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.

USP14 promotes tryptophan metabolism and immune suppression by stabilizing IDO1 in colorectal cancer.

Indoleamine 2,3 dioxygenase 1 (IDO1) is an attractive target for cancer immunotherapy. However, IDO1 inhibitors have shown disappointing therapeutic efficacy in clinical trials, mainly because of the activation of the aryl hydrocarbon receptor (AhR). Here, we show a post-transcriptional regulatory mechanism of IDO1 regulated by a proteasome-associated deubiquitinating enzyme, USP14, in colorectal cancer (CRC). Overexpression of USP14 promotes tryptophan metabolism and T-cell dysfunction by stabilizing the IDO1 protein. Knockdown of USP14 or pharmacological targeting of USP14 decreases IDO1 expression, reverses suppression of cytotoxic T cells, and increases responsiveness to anti-PD-1 in a MC38 syngeneic mouse model. Importantly, suppression of USP14 has no effects on AhR activation induced by the IDO1 inhibitor. These findings highlight a relevant role of USP14 in post-translational regulation of IDO1 and in the suppression of antitumor immunity, suggesting that inhibition of USP14 may represent a promising strategy for CRC immunotherapy.

Protein phosphatase 1 regulatory inhibitor subunit 14C promotes triple-negative breast cancer progression via sustaining inactive glycogen synthase kinase 3 beta.

Triple-negative breast cancer (TNBC) is fast-growing and highly metastatic with the poorest prognosis among the breast cancer subtypes. Inactivation of glycogen synthase kinase 3 beta (GSK3ß) plays a vital role in the aggressiveness of TNBC; however, the underlying mechanism for sustained GSK3ß inhibition remains largely unknown. Here, we find that protein phosphatase 1 regulatory inhibitor subunit 14C (PPP1R14C) is upregulated in TNBC and relevant to poor prognosis in patients. Overexpression of PPP1R14C facilitates cell proliferation and the aggressive phenotype of TNBC cells, whereas the depletion of PPP1R14C elicits opposite effects. Moreover, PPP1R14C is phosphorylated and activated by protein kinase C iota (PRKCI) at Thr73. p-PPP1R14C then represses Ser/Thr protein phosphatase type 1 (PP1) to retain GSK3ß phosphorylation at high levels. Furthermore, p-PPP1R14C recruits E3 ligase, TRIM25, toward the ubiquitylation and degradation of non-phosphorylated GSK3ß. Importantly, the blockade of PPP1R14C phosphorylation inhibits xenograft tumorigenesis and lung metastasis of TNBC cells. These findings provide a novel mechanism for sustained GSK3ß inactivation in TNBC and suggest that PPP1R14C might be a potential therapeutic target.

Nicotine-mediated OTUD3 downregulation inhibits VEGF-C mRNA decay to promote lymphatic metastasis of human esophageal cancer.

Nicotine addiction and the occurrence of lymph node spread are two major significant factors associated with esophageal cancer's poor prognosis; however, nicotine's role in inducing lymphatic metastasis of esophageal cancer remains unclear. Here we show that OTU domain-containing protein 3 (OTUD3) is downregulated by nicotine and correlates with poor prognosis in heavy-smoking esophageal cancer patients. OTUD3 directly interacts with ZFP36 ring finger protein (ZFP36) and stabilizes it by inhibiting FBXW7-mediated K48-linked polyubiquitination. ZFP36 binds with the VEGF-C 3-'UTR and recruits the RNA degrading complex to induce its rapid mRNA decay. Downregulation of OTUD3 and ZFP36 is essential for nicotine-induced VEGF-C production and lymphatic metastasis in esophageal cancer. This study establishes that the OTUD3/ZFP36/VEGF-C axis plays a vital role in nicotine addiction-induced lymphatic metastasis, suggesting that OTUD3 may serve as a prognostic marker, and induction of the VEGF-C mRNA decay might be a potential therapeutic strategy against human esophageal cancer.

Nicotine-Induced ILF2 Facilitates Nuclear mRNA Export of Pluripotency Factors to Promote Stemness and Chemoresistance in Human Esophageal Cancer.

Balancing mRNA nuclear export kinetics with its nuclear decay is critical for mRNA homeostasis control. How this equilibrium is aberrantly disrupted in esophageal cancer to acquire cancer stem cell properties remains unclear. Here we find that the RNA-binding protein interleukin enhancer binding factor 2 (ILF2) is robustly upregulated by nicotine, a major chemical component of tobacco smoke, via activation of JAK2/STAT3 signaling and significantly correlates with poor prognosis in heavy-smoking patients with esophageal cancer. ILF2 bound the THO complex protein THOC4 as a regulatory cofactor to induce selective interactions with pluripotency transcription factor mRNAs to promote their assembly into export-competent messenger ribonucleoprotein complexes. ILF2 facilitated nuclear mRNA export and inhibited hMTR4-mediated exosomal degradation to promote stabilization and expression of SOX2, NANOG, and SALL4, resulting in enhanced stemness and tumor-initiating capacity of esophageal cancer cells. Importantly, inducible depletion of ILF2 significantly increased the therapeutic efficiency of cisplatin and abrogated nicotine-induced chemoresistance in vitro and in vivo. These findings reveal a novel role of ILF2 in nuclear mRNA export and maintenance of cancer stem cells and open new avenues to overcome smoking-mediated chemoresistance in esophageal cancer. SIGNIFICANCE: This study defines a previously uncharacterized role of nicotine-regulated ILF2 in facilitating nuclear mRNA export to promote cancer stemness, suggesting a potential therapeutic strategy against nicotine-induced chemoresistance in esophageal cancer.

ALG3 contributes to stemness and radioresistance through regulating glycosylation of TGF-ß receptor II in breast cancer.

BACKGROUND: Radiotherapy is a conventional and effective local treatment for breast cancer. However, residual or recurrent tumors appears frequently because of radioresistance. Novel predictive marker and the potential therapeutic targets of breast cancer radioresistance needs to be investigated. METHODS: In this study, we screened all 10 asparagine-linked glycosylation (ALG) members in breast cancer patients' samples by RT-PCR. Cell viability after irradiation (IR) was determined by CCK-8 assay and flow cytometry. The radiosensitivity of cell lines with different ALG3 expression was determined with the colony formation assay by fitting the multi-target single hit model to the surviving fractions. Cancer stem-like traits were assessed by RT-PCR, Western blot, and flow cytometry. The mechanisms of ALG3 influencing radiosensitivity was detected by Western blot and immunoprecipitation. And the effect of ALG3 on tumor growth after IR was verified in an orthotopic xenograft tumor models. The association of ALG3 with prognosis of breast cancer patients was confirmed by immunohistochemistry. RESULTS: ALG3 was the most significantly overexpressing gene among ALG family in radioresistant breast cancer tissue. Overexpression of ALG3 predicted poor clinicopathological characteristics and overall survival (OS), and early local recurrence-free survival (LRFS) in breast cancer patients. Upregulating ALG3 enhanced radioresistance and cancer stemness in vitro and in vivo. Conversely, silencing ALG3 increased the radiosensitivity and repressed cancer stemness in vitro, and more importantly inhibition of ALG3 effectively increased the radiosensitivity of breast cancer cells in vivo. Mechanistically, our results further revealed ALG3 promoted radioresistance and cancer stemness by inducing glycosylation of TGF-ß receptor II (TGFBR2). Importantly, both attenuation of glycosylation using tunicamycin and inhibition of TGFBR2 using LY2109761 differentially abrogated the stimulatory effect of ALG3 overexpression on cancer stemness and radioresistance. Finally, our findings showed that radiation played an important role in preventing early recurrence in breast cancer patients with low ALG3 levels, but it had limited efficacy in ALG3-overexpressing breast cancer patients. CONCLUSION: Our results suggest that ALG3 may serve as a potential radiosensitive marker, and an effective target to decrease radioresistance by regulating glycosylation of TGFBR2 in breast cancer. For patients with low ALG3 levels, radiation remains an effective mainstay therapy to prevent early recurrence in breast cancer.

BOP1 confers chemoresistance of triple-negative breast cancer by promoting CBP-mediated ß-catenin acetylation.

Chemoresistance is a major obstacle to the treatment of triple-negative breast cancer (TNBC), which has a poor prognosis. Increasing evidence has demonstrated the essential role of cancer stem cells (CSCs) in the process of TNBC chemoresistance. However, the underlying mechanism remains unclear. In the present study, we report that block of proliferation 1 (BOP1) serves as a key regulator of chemoresistance in TNBC. BOP1 expression was significantly upregulated in chemoresistant TNBC tissues, and high expression of BOP1 correlated with shorter overall survival and relapse-free survival in patients with TNBC. BOP1 overexpression promoted, while BOP1 downregulation inhibited the drug resistance and CSC-like phenotype of TNBC cells in vitro and in vivo. Moreover, BOP1 activated Wnt/ß-catenin signaling by increasing the recruitment of cyclic AMP response element-binding protein (CBP) to ß-catenin, enhancing CBP-mediated acetylation of ß-catenin, and increasing the transcription of downstream stemness-related genes CD133 and ALDH1A1. Notably, treating with the ß-catenin/CBP inhibitor PRI-724 induced an enhancement of chemotherapeutic response of paclitaxel in BOP1-overexpressing TNBC cells. These findings indicate that BOP1 is involved in chemoresistance development and might serve as a prognostic marker and therapeutic target in TNBC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Actin-like protein 6A/MYC/CDK2 axis confers high proliferative activity in triple-negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with high proliferative activity. TNBC tumors exhibit elevated MYC expression and altered expression of MYC regulatory genes, which are associated with tumor progression and poor prognosis; however, the underlying mechanisms by which MYC retains its high expression and mediates TNBC tumorigenesis require further exploration. METHODS: ACTL6A regulation of MYC and its target gene, CDK2, was defined using Co-IP, mass spectrometry and ChIP assays. To study the role of ACTL6A in TNBC, we performed soft-agar, colony formation, flow cytometry and tumor formation in nude mice. CDK2 inhibitor and paclitaxel were used in testing combination therapy in vitro and in vivo. RESULTS: ACTL6A bound MYC to suppress glycogen synthase kinase 3 beta (GSK3ß)-induced phosphorylation on MYC T58, which inhibited ubiquitination of MYC and stabilized it. Moreover, ACTL6A promoted the recruitment of MYC and histone acetyltransferase KAT5 on CDK2 promoters, leading to hyperactivation of CDK2 transcription. ACTL6A overexpression promoted, while silencing ACTL6A suppressed cell proliferation and tumor growth in TNBC cells in vitro and in vivo, which was dependent on MYC signaling. Furthermore, co-therapy with paclitaxel and CDK2 inhibitor showed synergistic effects in tumor suppression. Notably, ACTL6A/MYC/CDK2 axis was specifically up-regulated in TNBC and high expression of ACTL6A was correlated to shorter survival in patients with TNBC. CONCLUSIONS: These findings reveal a novel mechanism by which ACTL6A prolongs the retention of MYC in TNBC and suggest that pharmacological targeting ACTL6A/MYC/CDK2 axis might have therapeutic potential in patients with TNBC.

TRIB3 confers radiotherapy resistance in esophageal squamous cell carcinoma by stabilizing TAZ.

Radioresistance becomes the major obstacle to reduce tumor recurrence and improve prognosis in the treatment of esophageal squamous cell carcinoma (ESCC). Thus new strategies for radioresistant ESCC are urgently needed. Herein, we reported that tribbles pseudokinase 3 (TRIB3) serves as a key regulator of radioresistance in ESCC. TRIB3 is overexpressed in ESCC tissues and cell lines. High expression of TRIB3 significantly correlates with poor radiotherapy response and prognosis in ESCC patients. Upregulation of TRIB3 in ESCC cells conferred radioresistance in vitro and in vivo by interacting with TAZ thus impeding ß-TrCP-mediated TAZ ubiquitination and degradation. Conversely, silencing TRIB3 sensitized ESCC cells to ionizing radiation. More importantly, TRIB3 was significantly correlated with TAZ activation in ESCC biopsies, and patients with high expression of both TRIB3 and TAZ suffered the worst radiotherapy response and survival. Our study uncovers the critical mechanism of ESCC resistance to radiotherapy, and provides a new pharmacological opportunity for developing a mechanism-based strategy to eliminate radioresistant ESCC in clinical practice.

CHAF1B induces radioresistance by promoting DNA damage repair in nasopharyngeal carcinoma.

BACKGROUND: Radiotherapy is the main treatment for nasopharyngeal carcinoma (NPC); however radioresistance restricts its efficacy. Therefore, new molecular regulators are required to improve the radiosensitivity of NPC. Chromatin assembly factor 1 subunit B (CHAF1B) plays a role in DNA synthesis and repair, and participates in the progression of various malignancies. However, the expression and function of CHAF1B in NPC is unclear. METHODS: The expression of CHAF1B was determined using real-time PCR and western blotting. CHAF1B expression in 160 human NPC tissue samples was evaluated using immunochemistry (IHC). The correlations between CHAF1B expression and NPC clinicopathological features were determined. The effect of CHAF1B on the radiosensitivity of NPC cells was detected using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and colony formation assays. Apoptosis rates were analyzed using flow cytometry. A nude mouse subcutaneous xenograft model and living fluorescence imaging were applied to evaluate tumor regression in vivo. The molecular mechanisms of radioresistance were confirmed by bioinformatics analysis and detection of phosphorylated H2A histone family member X (γH2AX) foci. RESULTS: Significantly increased CHAF1B levels were observed in NPC tissues, which correlated positively with radioresistance and poor prognosis. In addition, CHAF1B was upregulated in radioresistant NPC cell lines. Overexpression of CHAF1B reduced, while silencing of CHAF1B enhanced, the radiosensitivity of NPC cells in vitro and in vivo. Mechanistically, CHAF1B inhibited NPC cell apoptosis by promoting DNA damage repair. Finally, the DNA-dependent protein kinase (DNA-PK) pathway was observed to be essential for CHAF1B promotion of DNA damage repair-mediated radioresistance. CONCLUSION: The results suggested CHAF1B enhances radioresistance by promoting DNA damage repair and inhibiting cell apoptosis, in a DNA-PK pathway-dependent manner. CHAF1B may serve as a novel factor for predicting radiorsensitivity. Besides, DNA-dependent protein kinase inhibitor could serve as a radiosensitizer for patients with NPC and high CHAF1B expression.

Epigenetic silencing of SALL2 confers tamoxifen resistance in breast cancer.

Resistance to tamoxifen is a clinically major challenge in breast cancer treatment. Although downregulation of estrogen receptor-alpha (ERα) is the dominant mechanism of tamoxifen resistance, the reason for ERα decrease during tamoxifen therapy remains elusive. Herein, we reported that Spalt-like transcription factor 2 (SALL2) expression was significantly reduced during tamoxifen therapy through transcription profiling analysis of 9 paired primary pre-tamoxifen-treated and relapsed tamoxifen-resistant breast cancer tissues. SALL2 transcriptionally upregulated ESR1 and PTEN through directly binding to the DNA promoters. By contrast, silencing SALL2 induced downregulation of ERα and PTEN and activated the Akt/mTOR signaling, resulting in estrogen-independent growth and tamoxifen resistance in ERα-positive breast cancer. Furthermore, hypermethylation of SALL2 promoter was found in tamoxifen-resistant breast cancer. Importantly, in vivo experiments showed that DNA methyltransferase inhibitor-mediated SALL2 restoration resensitized tamoxifen-resistant breast cancer to tamoxifen therapy. These findings shed light on the mechanism of SALL2 in regulation of ER and represent a potential clinical signature that can be used to categorize breast cancer patients who may benefit from co-therapy with tamoxifen and DNMT inhibitor.

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.

Jade family PHD finger 3 (JADE3) increases cancer stem cell-like properties and tumorigenicity in colon cancer.

Jade family PHD finger 3 (JADE3) plays a role in inducing histone acetylation during transcription, and is involved in the progression of several human cancers; however, its role in colon cancer remains unclear. Herein, we found that JADE3 was markedly upregulated in colon cancer tissues and significantly correlated with cancer progression, and predicted shorter patient survival. Further, JADE3 was expressed much higher in colon cancer cell lines that are enriched with a stem-like signature. Overexpression of JADE3 increased, while silencing JADE3 reduced cancer stem cell-like traits in colon cancer cells in vitro and in vivo. Importantly, silencing of JADE3 strongly impaired the tumor initiating capacity of colon cancer cells in vivo. Furthermore, JADE3 interacted with the promoters of colon stem cell marker LGR5 and activated its transcription, by increasing the occupancy of p300 acetyltransferase and histone acetylation on the promoters. Finally, we found that JADE3 expression was substantially induced by Wnt/ß-catenin signaling. These findings suggest an oncogenic role of JADE3 by regulating cancer stem cell-like traits in the colon cancer, and therefore JADE3 might be a potential therapeutic target for the treatment of colon cancer.

Minocycline enhances mitomycin C-induced cytotoxicity through down-regulating ERK1/2-mediated Rad51 expression in human non-small cell lung cancer cells.

Minocycline is a semisynthetic tetracycline derivative; it has anti-inflammatory and anti-cancer effects distinct from its antimicrobial function. However, the molecular mechanism of minocycline-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination and high levels of Rad51 expression are observed in chemo- or radioresistant carcinomas. Our previous studies have shown that the MKK1/2-ERK1/2 signal pathway maintains the expression of Rad51 in NSCLC cells. In this study, minocycline treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1975. Treatment with minocycline decreased Rad51 mRNA and protein levels through MKK1/2-ERK1/2 inactivation. Furthermore, expression of constitutively active MKK1 (MKK1-CA) vectors significantly rescued the decreased Rad51 protein and mRNA levels in minocycline-treated NSCLC cells. However, combined treatment with MKK1/2 inhibitor U0126 and minocycline further decreased the Rad51 expression and cell viability of NSCLC cells. Knocking down Rad51 expression by transfection with small interfering RNA of Rad51 enhanced the cytotoxicity and cell growth inhibition of minocycline. Mitomycin C (MMC) is typically used as a first or second line regimen to treat NSCLC. Compared to a single agent alone, MMC combined with minocycline resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-ERK1/2, and reduced Rad51 protein levels. Overexpression of MKK1-CA or Flag-tagged Rad51 could reverse the minocycline and MMC-induced synergistic cytotoxicity. These findings may have implications for the rational design of future drug regimens incorporating minocycline and MMC for the treatment of NSCLC.

Resveratrol Enhances Etoposide-Induced Cytotoxicity through Down-Regulating ERK1/2 and AKT-Mediated X-ray Repair Cross-Complement Group 1 (XRCC1) Protein Expression in Human Non-Small-Cell Lung Cancer Cells.

Etoposide (VP-16), a topoisomerase II inhibitor, is an effective anti-cancer drug used for the treatment of non-small-cell lung cancer (NSCLC). Resveratrol is a naturally occurring polyphenolic compound that has been proved to have anti-cancer activity. XRCC1 is an important scaffold protein involved in base excision repair that is regulated by ERK1/2 and AKT signals and plays an important role in the development of lung cancer. However, the role of ERK1/2 and AKT-mediated XRCC1 expression in etoposide treatment alone or combined with resveratrol-induced cytotoxicity in NSCLC cells has not been identified. In this study, etoposide treatment increased XRCC1 mRNA and protein expression through AKT and ERK1/2 activation in two NSCLC cells, H1703 and H1975. Knockdown of XRCC1 in NSCLC cells by transfection of XRCC1 siRNA or inactivation of ERK1/2 and AKT resulted in enhancing cytotoxicity and cell growth inhibition induced by etoposide. Resveratrol inhibited the expression of XRCC1 and enhanced the etoposide-induced cell death and anti-proliferation effect in NSCLC cells. Furthermore, transfection with constitutive active MKK1 or AKT vectors could rescue the XRCC1 protein level and also the cell survival suppressed by co-treatment with etoposide and resveratrol. These findings suggested that down-regulation of XRCC1 expression by resveratrol can enhance the chemosensitivity of etoposide in NSCLC cells.

Down-regulation of ERK1/2 and AKT-mediated X-ray repair cross-complement group 1 protein (XRCC1) expression by Hsp90 inhibition enhances the gefitinib-induced cytotoxicity in human lung cancer cells.

Gefitinib (Iressa(R), ZD1839) is a selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that blocks growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT signaling activation. It has been shown that inhibition of Hsp90 function can enhance antitumor activity of EGFR-TKI. XRCC1 is an important scaffold protein in base excision repair, which could be regulated by ERK1/2 and AKT pathways. However, the role of ERK1/2 and AKT-mediated XRCC1 expression in gefitinib alone or combination with an Hsp90 inhibitor-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. In this study, gefitinib treatment decreased XRCC1 mRNA and protein expression through ERK1/2 and AKT inactivation in two NSCLC cells, A549 and H1975. Knocking down XRCC1 expression by transfection with small interfering RNA of XRCC1 enhanced the cytotoxicity and cell growth inhibition of gefitinib. Combining treatment of gefitinib with an Hsp90 inhibitor resulted in enhancing the reduction of XRCC1 protein and mRNA levels in gefitinib-exposed A549 and H1975 cells. Compared to a single agent alone, gefitinib combined with an Hsp90 inhibitor resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells. Furthermore, transfection with constitutive active MKK1 or AKT vectors rescued the XRCC1 protein level as well as the cell survival suppressed by an Hsp90 inhibitor and gefitinib. These findings suggested that down-regulation of XRCC1 can enhance the sensitivity of gefitinib for NSCLC cells.

Down-regulation of MSH2 expression by Hsp90 inhibition enhances cytotoxicity affected by tamoxifen in human lung cancer cells.

The anti-estrogen tamoxifen has been used worldwide as an adjuvant hormone therapeutic agent in the treatment of breast cancer. However, the molecular mechanism of tamoxifen-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Human MutS homolog 2 (MSH2), a crucial element of the highly conserved DNA mismatch repair system, and expression of MSH2 have been down-regulated by Hsp90 function inhibition in human lung cancer. Therefore, in this study, we examined whether MSH2 plays a role in the tamoxifen and Hsp90 inhibitor-induced cytotoxic effect on NSCLC cells. The results showed that treatment with tamoxifen increased MSH2 mRNA and protein levels. The combination treatment with PI3K inhibitors (LY294002 or wortmannin) or knockdown AKT expression by specific small interfering RNA could decrease tamoxifen-induced MSH2 expression. Both knocking down MSH2 expression and co-treatment of PI3K inhibitors enhanced the cytotoxicity and cell growth inhibition of tamoxifen. Compared to a single agent alone, tamoxifen combined with an Hsp90 inhibitor resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced MSH2 expression. These findings may have implications for the rational design of future drug regimens incorporating tamoxifen and Hsp90 inhibitors for the treatment of NSCLC.