Knockdown of KLF5 promotes cisplatin‐induced cell apoptosis via regulating DNA damage checkpoint proteins in non‐small cell lung cancer (original) (raw)
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Targeted reduction of KLF6-SV1 restores chemotherapy sensitivity in resistant lung adenocarcinoma
Lung Cancer, 2009
Kruppel-like factor 6 splice variant 1 (KLF6-SV1) is an oncogenic splice variant of the KLF6 tumor suppressor gene that is specifically overexpressed in a number of human cancers. Previously, we have demonstrated that increased expression of KLF6-SV1 is associated with decreased survival in lung adenocarcinoma patient samples and that targeted reduction of KLF6-SV1 using siRNA induced apoptosis both alone and in combination with the chemotherapeutic drug cisplatin. Here, we demonstrate that chemoresistant lung cancer cells express increased levels of KLF6-SV1. Furthermore, targeted reduction of KLF6-SV1 using RNA interference restores chemotherapy sensitivity to lung cancer cells both in culture and in vivo through induction of apoptosis. Conversely, overexpression of KLF6-SV1 resulted in a marked reduction in chemotherapy sensitivity in a tumor xenograft model. Combined, these findings highlight a functional role for the KLF6-SV1 splice variant in the regulation of chemotherapy response in lung cancer and could provide novel insight into lung cancer therapy.
A Functional Role for KLF6SV1 in Lung Adenocarcinoma Prognosis and Chemotherapy Response
Kruppel-like factor 6 (KLF6) is a tumor suppressor gene that is functionally inactivated in human cancer by loss of heterozygosity, somatic mutation, decreased expression, and increased alternative splicing into an oncogenic splice variant, KLF6-SV1. Here we show that increased expression of KLF6-SV1 is associated with decreased survival in patients with lung adenocarcinoma. In addition, KLF6-SV1 is a novel antiapoptotic protein in lung cancer cell lines, and targeted reduction of KLF6-SV1 using siRNA induces apoptosis both alone and in combination with the chemotherapeutic drug cisplatin. Together, these findings highlight a critical role for KLF6-SV1 in lung cancer, and show a potential novel therapeutic strategy for the treatment of lung cancer. [Cancer Res 2008;68(4):965-70] Requests for reprints: Goutham Narla, Mount
KLF6 degradation after apoptotic DNA damage
2006
Krüppel-like factor 6 (KLF6) is a cancer gene (www.sanger.ac.uk/genetics/CGP/Census/). Here, we demonstrate that KLF6 protein is rapidly degraded when apoptosis is induced via the intrinsic pathway by cisplatin, adriamycin, or UVB irradiation in multiple cell lines (HCT116, SW40, HepG2, PC3-M, Skov3, NIH-3T3, 293T, GM09706, and MEF, IMR-90). KLF6 degradation occurred in the presence or absence of p53, was associated with ubiquitination, mediated by the proteasome (half-life 16 min, unstimulated), and independent of caspases and calpain. KLF6 was unchanged by apoptosis via the extrinsic/death-receptor pathway. Deregulation of KLF6 stability may alter its tumor suppressor function and/or the response of tumors to chemotherapeutics.
The Kruppel-like transcription factors (KLFs) 4 and 5 (KLF4/5) are coexpressed in mouse embryonic stem cells, where they function redundantly to maintain pluripotency. In mammary carcinoma, KLF4/5 can each impact the malignant phenotype, but potential linkages to drug resistance remain unclear. In primary human breast cancers, we observed a positive correlation between KLF4/5 transcript abundance, particularly in the human epidermal growth factor receptor 2 (HER2)-enriched subtype. Furthermore, KLF4/5 protein was rapidly upregulated in human breast cancer cells following treatment with the HER2/epidermal growth factor receptor inhibitor, lapatinib. In addition, we observed a positive correlation between these factors in the primary tumors of genetically engineered mouse models (GEMMs). In particular, the levels of both factors were enriched in the basal-like tumors of the C3(1) TAg (SV40 large T antigen transgenic mice under control of the C3(1)/prostatein promoter) GEMM. Using tumor cells derived from this model as well as human breast cancer cells, suppression of KLF4 and/or KLF5 sensitized HER2 -overexpressing cells to lapatinib. Indicating cooperativity, greater effects were observed when both genes were depleted. KLF4/5-deficient cells had reduced basal mRNA and protein levels of the anti-apoptotic factors myeloid cell leukemia 1 (MCL1) and B-cell lymphoma-extra large (BCL-XL). Moreover, MCL1 was upregulated by lapatinib in a KLF4/5-dependent manner, and enforced expression of MCL1 in KLF4/5-deficient cells restored drug resistance. In addition, combined suppression of KLF4/5 in cultured tumor cells additively inhibited anchorage-independent growth, resistance to anoikis and tumor formation in immunocompromised mice. Consistent with their cooperative role in drug resistance and other malignant properties, KLF4/5 levels selectively stratified human HER2-enriched breast cancer by distant metastasis-free survival. These results identify KLF4 and KLF5 as cooperating protumorigenic factors and critical participants in resistance to lapatinib, furthering the rationale for combining anti-MCL1/BCL-XL inhibitors with conventional HER2-targeted therapies.
Targeting NF-κB-mediated inflammatory pathways in cisplatin-resistant NSCLC
Lung Cancer, 2019
The majority of patients with non-small cell lung cancer (NSCLC) present with advanced stage disease, at which time chemotherapy is usually the most common treatment option. While somewhat effective, patients treated with platinum-based regimens will eventually develop resistance, with others presenting with intrinsic resistance. Multiple pathways have been implicated in chemo-resistance, however the critical underlying mechanisms have yet to be elucidated. The aim of this project was to determine the role of inflammatory mediators in cisplatin-resistance in NSCLC.
Oncogene, 2008
Although Kruppel-like factor 5 (KLF5) is a transcription factor that has been implicated in pathways critical to carcinogenesis, controversy persists as to whether it functions as a tumor suppressor or as an oncogene. Here, we describe a novel role for KLF5 in a p53-independent apoptotic pathway. Using RNA-interference technology, we show that cells deficient in KLF5 have increased sensitivity to DNA damage, regardless of p53 status. Both p53 and p53-dependent factors are unaffected by KLF5 depletion. Instead, the apoptotic phenotype consequent to damage is associated with reduced bad phosphorylation, and downregulation of Pim1. Consistently, transfection of wild-type Pim1 is sufficient to rescue this phenotype. Previous data have shown a number of putative Sp1binding consensus sequences on the Pim1 promoter.
Strategic targeting of the PI3K–NFκB axis in cisplatin-resistant NSCLC
Cancer Biology & Therapy, 2014
Chemoresistance is a major therapeutic challenge to overcome in NSCLC, in order to improve the current survival rates of <15% at 5 years. We and others have shown increased PI3K signalling in NSCLC to be associated with a more aggressive disease, and a poorer prognosis. In this study, targeted inhibition of three strategic points of the PI3K-NFκB axis was carried out with the aim of exploiting vulnerabilities in cisplatin resistant NSCLC cells. Cisplatin resistant cell lines were previously generated through prolonged exposure to the drug. Expression of PI3K and NFκB pathway related genes were compared between cisplatin resistant cells and their matched parent cells using a gene expression array, qRT-PCR, DNA sequencing, Western blot and immunofluorescence. Targeted inhibition was carried out using GDC-0980, a dual PI3K-mTOR inhibitor currently in Phase II clinical trials in NSCLC, and DHMEQ, an inhibitor of NFB translocation which has been used extensively both in vitro and in vivo. Effects of the two inhibitors were assessed by BrdU proliferation assay and multiparameter viability assay. NFKBIA was shown to be 12 fold overexpressed in cisplatin resistant cells, with no mutations present in exons 3, 4 or 5 of the gene. Corresponding overexpression of IκBα was also observed. Treatment with DHMEQ (but not GDC-0980) led to significantly enhanced effects on viability and proliferation in cisplatin resistant cells compared to parent cells. We conclude that NFκB inhibition represents a more promising strategy than PI3K-mTOR inhibition for treatment in the chemoresistance setting in NSCLC.
The role of KLF6 and its splice variants in cancer therapy
Drug Resistance Updates, 2009
The Krüppel-like zinc finger transcription factor (KLF6) gene encodes a family of proteins generated through alternative splicing involved in the regulation of cancer development and progression. Alternative splicing of the KLF6 gene results in the production of at least four alternatively spliced isoforms, two of which are extensively discussed in this review. The full length form of the KLF6 gene is a tumor suppressor gene that is frequently inactivated by loss of heterozygozity (LOH), somatic mutation, and/or decreased expression in human cancer. While the exact mechanisms underlying KLF6's tumor suppressor roles are not completely known, a number of highly relevant, overlapping pathways have been described: transactivation of p21 in a p53-independent manner, reduction of cyclin D1/cdk4 complexes via interaction with cyclin D1, inhibition of c-Jun proto-oncoprotein activities, decreased VEGF expression, and induction of apoptosis. Kruppel-like factor 6 splice variant 1 (KLF6-SV1) is an oncogenic splice variant of the KLF6 tumor suppressor gene that is specifically overexpressed in a number of human cancers. Increased KLF6-SV1 expression is associated with poor prognosis in prostate, lung, and ovarian cancer. Furthermore, KLF6-SV1 has been shown to be biologically active, antagonizing the tumor suppressor function of KLF6 and promoting tumor growth and dissemination in both ovarian and prostate cancer models. In addition, a common germline polymorphism in the KLF6 gene associated with increased prostate cancer risk in a large multi-institutional study of 3411 men results in increased expression of KLF6-SV1. Furthermore, recent studies have demonstrated that targeted reduction of KLF6-SV1 results in the induction of spontaneous apoptosis in cell culture, synergizes with chemotherapeutic agents like cisplatin, and results in significant tumor regression in vivo. Combined, these data make the KLF6 gene family a compelling therapeutic target for both the treatment of localized as well as metastatic cancer.
DNA Repair Response Modulation Potentiates Low Dose Cisplatin Effects in HeLa Cells
Oncomedicine, 2018
Background: Cisplatin is considered as the crucial regimen of widely prescribed chemotherapy treatment to many cancer types. However, incessant increasing dose of cisplatin against many cancers including cervical cancer is known for drug resistance and side effects like nephrotoxicity and ototoxicity. Methods: To address above problems, this paper reports using ATM kinase inhibitor (KU55933) and siRNA DNA ligase III to alter the DNA repair response in vitro low dose cisplatin treated HeLa cells. Here, authors performed cell proliferation, MTT cell cytotoxicity, annexin V/PI staining flow cytometery and PI stained cell cycle assays to substantiate observations. Results: This paper reports that low dose cisplatin (10 µM) can show better effects in vitro growing HeLa cells in combination with KU55933. However, we included siRNA DNA ligase III along with KU55933, but data suggest that the level of DNA ligase III protein are not changed due to treatment with siRNA DNA ligase III as shown by immunocytochemistry assay. Even though, level of DNA ligase III is not altered in the present finding, use of KU55933 as combinatorial option with low dose cisplatin produce significant anti-proliferation effects (up to 35%) and also loss of cell viability including apoptotic cell death (up to 30%) in treated HeLa cells. Conclusion: This study suggests that KU55933 in combination with low dose cisplatin may be a good option to bring anti-proliferation and loss of viability in HeLa cells and may be explored in other cancer cell lines as well.
Balance between MKK6 and MKK3 Mediates p38 MAPK Associated Resistance to Cisplatin in NSCLC
PLoS ONE, 2011
The p38 MAPK signaling pathway has been proposed as a critical mediator of the therapeutic effect of several antitumor agents, including cisplatin. Here, we found that sensitivity to cisplatin, in a system of 7 non-small cell lung carcinoma derived cell lines, correlated with high levels of MKK6 and marked activation of p38 MAPK. However, knockdown of MKK6 modified neither the response to cisplatin nor the activation of p38 MAPK. Deeper studies showed that resistant cell lines also displayed higher basal levels of MKK3. Interestingly, MKK3 knockdown significantly decreased p38 phosphorylation upon cisplatin exposure and consequently reduced the response to the drug. Indeed, cisplatin poorly activated MKK3 in resistant cells, while in sensitive cell lines MKK3 showed the opposite pattern in response to the drug. Our data also demonstrate that the low levels of MKK6 expressed in resistant cell lines are the consequence of high basal activity of p38 MAPK mediated by the elevated levels of MKK3. This finding supports the existence of a regulatory mechanism between both MAPK kinases through their MAPK. Furthermore, our results were also mirrored in head and neck carcinoma derived cell lines, suggesting our observations boast a potential universal characteristic in cancer resistance of cisplatin. Altogether, our work provides evidence that MKK3 is the major determinant of p38 MAPK activation in response to cisplatin and, hence, the resistance associated with this MAPK. Therefore, these data suggest that the balance between both MKK3 and MKK6 could be a novel mechanism which explains the cellular response to cisplatin. Citation: Galan-Moya EM, de la Cruz-Morcillo MA, Valero ML, Callejas-Valera JL, Melgar-Rojas P, et al. (2011) Balance between MKK6 and MKK3 Mediates p38 MAPK Associated Resistance to Cisplatin in NSCLC. PLoS ONE 6(12): e28406.