Debarati Mukherjee

KLF8 and FAK cooperatively enrich the active MMP14 on the cell surface required for the metastatic progression of breast cancer

Krüppel-like factor 8 (KLF8) regulates critical gene transcription associated with cancer. The underlying mechanisms, however, remain largely unidentified. We have recently demonstrated that KLF8 expression enhances the activity but not expression of matrix metalloproteinase-2 (MMP2), the target substrate of MMP14. Here, we report a novel KLF8 to MMP14 signaling that promotes human breast cancer invasion and metastasis. Using cell lines for inducible expression and knockdown of KLF8, we demonstrate that KLF8 promotes MMP14 expression at the transcriptional level. Knocking down KLF8 expression inhibited the breast cancer cell invasion both in vitro and in vivo as well as the lung metastasis in mice, which could be rescued by ectopic expression of MMP14. Promoter reporter assays and oligonucleotide and chromatin immunoprecipitations determined that KLF8 activates the human MMP14 gene promoter by both directly acting on the promoter and indirectly via promoting the nuclear translocation of β-catenin, the expression of T-cell factor-1 (TCF1) and subsequent activation of the promoter by the β-catenin/TCF1 complex. Inhibition of focal adhesion kinase (FAK) using pharmacological inhibitor, RNA interference or knockout showed that the cell surface presentation of active MMP14 downstream of KLF8 depends on FAK expression and activity. Taken together, this work identified novel signaling mechanisms by which KLF8 and FAK work together to promote the extracellular activity of MMP14 critical for breast cancer metastasis.

A novel role of Kruppel-like factor 8 in DNA repair in breast cancer cells

Background: KLF8 is a cancer-promoting transcription factor. Results: KLF8 inhibits DNA damage in breast cancer cells. Conclusion: KLF8 is a novel effector of the PARP-1 and DNA-PK DNA damage response pathways. Significance: KLF8 could be targeted for chemosensitizing therapy. Krüppel-like factor 8 (KLF8) regulates critical gene transcription and cellular events associated with cancer. However, the role of KLF8 in cancer remains largely unknown. Here, we report a surprisingly novel role for KLF8 in DNA repair in breast cancer cells. Comet, clonogenic, and WST-1 assays showed that KLF8 expression is required for protecting human breast cancer cells from doxorubicin-induced DNA damage and cell death. Western blotting indicated that overexpression of ectopic KLF8 attenuated the levels of the DNA damage marker γH2A.X in doxorubicin-treated PARP-1+/+ but not PARP-1−/− mouse embryonic fibroblasts, whereas the PARP-1-binding-defective KLF8 mutant failed to do so. Interestingly, in response to the DNA damage, KLF8 was phosphorylated by the DNA-dependent protein kinase catalytic subunit and, subsequently, SUMOylated by SUMO E3 ligases protein inhibitors of activated STAT (PIASs), which depends upon the interaction of KLF8 with DNA-dependent protein kinase catalytic subunit, PIASs, and PARP-1 as well as their enzymatic activities. Lastly, we show evidence that KLF8 was recruited to the DNA damage site. These results suggest a novel role and mechanism for KLF8 in the regulation of DNA repair and therapeutic resistance in breast cancer cells.

Identification of epithelial stromal interaction 1 as a novel effector downstream of Krüppel-like factor 8 in breast cancer invasion and metastasis

Krüppel-like factor 8 (KLF8) is a transcriptional factor critical for metastatic progression of breast cancer. Epithelial stromal interaction 1 (EPSTI1), a recently identified stromal fibroblast-induced gene in non-invasive breast cancer cells is highly overexpressed in invasive breast carcinomas. The function and regulation of EPSTI1, however, remain largely unknown. In this paper, we report a novel KLF8 to EPSTI1 signaling pathway in breast cancer. Using various expression analyses, we revealed a high co-overexpression of KLF8 and EPSTI1 in invasive human breast cancer cells and patient tumors. Ectopic overexpression of KLF8 in the non-invasive MCF-10A cells induced the EPSTI1 expression, whereas KLF8 knockdown from the invasive, MDA-MB-231 cells decreased the EPSTI1 expression. Promoter activation and binding analyses indicated that KLF8 promoted the EPSTI1 expression by directly acting on the EPSTI1 gene promoter. EPSTI1 knockdown dramatically reduced the KLF8-promoted MCF-10A cell invasion, and ectopic expression of EPSTI1 in the non-invasive MCF-7 cells is sufficient to induce the cell invasion. Experiments using nude mice demonstrated that the ectopic EPSTI1 granted the MCF-7 cells capability of both invasive growth in the breasts and metastasis to the lungs. Using co-immunoprecipitation coupled with mass spectrometry, we discovered that EPSTI1 interacts with the valosin-containing protein (VCP), resulting in the degradation of IκBα and subsequent activation of NF-κB in the nucleus. These findings suggest a novel KLF8 to EPSTI1 to VCP to NF-κB signaling mechanism potentially critical for breast cancer invasion and metastasis.

Krüppel-like factor 8 activates the transcription of C-X-C cytokine receptor type 4 to promote breast cancer cell invasion, transendothelial migration and metastasis.

Krüppel-like factor 8 (KLF8) has been strongly implicated in breast cancer metastasis. However, the underlying mechanisms remain largely unknown. Here we report a novel signaling from KLF8 to C-X-C cytokine receptor type 4 (CXCR4) in breast cancer. Overexpression of KLF8 in MCF-10A cells induced CXCR4 expression at both mRNA and protein levels, as determined by quantitative real-time PCR and immunoblotting. This induction was well correlated with increased Boyden chamber migration, matrigel invasion and transendothelial migration (TEM) of the cells towards the ligand CXCL12. On the other hand, knockdown of KLF8 in MDA-MB-231 cells reduced CXCR4 expression associated with decreased cell migration, invasion and TEM towards CXCL12. Histological and database mining analyses of independent cohorts of patient tissue microarrays revealed a correlation of aberrant co-elevation of KLF8 and CXCR4 with metastatic potential. Promoter analysis indicated that KLF8 directly binds and activates the human CXCR4 gene promoter. Interestingly, a CXCR4-dependent activation of focal adhesion kinase (FAK), a known upregulator of KLF8, was highly induced by CXCL12 treatment in KLF8-overexpressing, but not KLF8 deficient cells. This activation of FAK in turn induced a further increase in KLF8 expression. Xenograft studies showed that overexpression of CXCR4, but not a dominant-negative mutant of it, in the MDA-MB-231 cells prevented the invasive growth of primary tumor and lung metastasis from inhibition by knockdown of KLF8. These results collectively suggest a critical role for a previously unidentified feed-forward signaling wheel made of KLF8, CXCR4 and FAK in promoting breast cancer metastasis and shed new light on potentially more effective anti-cancer strategies.

Cerium Oxide Nanoparticles Sensitize Pancreatic Cancer to Radiation Therapy through Oxidative Activation of the JNK Apoptotic Pathway

Side effects of radiation therapy (RT) remain the most challenging issue for pancreatic cancer treatment. Cerium oxide nanoparticles (CONPs) are currently being tested in pre-clinical trials as an adjuvant to sensitize pancreatic cancer cells to RT and protect normal tissues from the harmful side effects. CONPs were not able to significantly affect RT-induced DNA damage in cancer cells, thereby ruling out sensitization through increased mitotic catastrophe. However, activation of c-Jun terminal kinase (JNK), a key driver of RT-induced apoptosis, was significantly enhanced by co-treatment with CONPs and RT in pancreatic cancer cells in vitro and human pancreatic tumors in nude mice in vivo compared to CONPs or RT treatment alone. Further, CONP-driven increase in RT-induced JNK activity was associated with a marked increase in Caspase 3/7 activation, indicative of apoptosis. We have previously shown that CONPs increase reactive oxygen species (ROS) production in cancer cells. ROS has been shown to drive the oxidation of thioredoxin 1 (TRX1) which results in the activation of apoptosis signaling kinase 1 (ASK1). The increase in ASK1 activation following the co-treatment with CONPs followed by RT suggests that the increased JNK activation is the result of increased TRX1 oxidation. The ability of CONPs to sensitize pancreatic cancer cells to RT was mitigated when the TRX1 oxidation was prevented by mutagenesis of a cysteine residue or when the JNK activation was blocked by an inhibitor. Taken together, these data demonstrate an important mechanism for CONPs in specifically killing cancer cells and provide novel insights into the utilization of CONPs as a radiosensitizer and therapeutic agent for pancreatic cancer.

Abstract 1626: Krüppel-like factor 8 activates the transcription of C-X-C cytokine receptor type 4 to promote breast cancer cell invasion, transendothelial migration and metastasis

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Kruppel-like factor 8 (KLF8) has been strongly implicated in breast cancer metastasis. However, the underlying mechanisms remain largely unknown. Here we report a novel signaling from KLF8 to C-X-C cytokine receptor type 4 (CXCR4) in breast cancer. Overexpression of KLF8 in MCF-10A cells induced CXCR4 expression at both mRNA and protein levels, as determined by quantitative real-time PCR and immunoblotting. This induction was well correlated with increased Boyden chamber migration, matrigel invasion and transendothelial migration (TEM) of the cells towards the ligand CXCL12. On the other hand, knockdown of KLF8 in MDA-MB-231 cells reduced CXCR4 expression associated with decreased cell migration, invasion and TEM towards CXCL12. Histological and database mining analyses of independent cohorts of patient tissue microarrays revealed a correlation of aberrant co-elevation of KLF8 and CXCR4 with metastatic potential. Promoter analysis indicated that KLF8 directly binds and activates the human CXCR4 gene promoter. Interestingly, a CXCR4-dependent activation of focal adhesion kinase (FAK), a known upregulator of KLF8, was highly induced by CXCL12 treatment in KLF8-overexpressing, but not KLF8 deficient cells. This activation of FAK in turns induced a further increase in KLF8 expression. Xenograft studies showed that overexpression of CXCR4, but not a dominant-negative mutant of it, in the MDA-MB-231 cells prevented the invasive growth of primary tumor and lung metastasis from inhibition by knockdown of KLF8. These results collectively suggest a critical role for a previously unidentified feed-forward signaling wheel made of KLF8, CXCR4 and FAK in promoting breast cancer metastasis and shed new light on potentially more effective anti-cancer strategies. Citation Format: Debarati Mukherjee, Heng Lu, Lin Yu, Satadru K. Lahiri, Tianshu Li, Jihe Zhao. Kruppel-like factor 8 activates the transcription of C-X-C cytokine receptor type 4 to promote breast cancer cell invasion, transendothelial migration and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1626.

Abstract 5161: Identification of epithelial stromal interaction 1 as a novel mediator of breast cancer cell invasion and metastasis downstream of Krüppel-like factor 8.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Epithelial stromal interaction 1 (EPSTI1) is a recently identified novel gene that is highly upregulated in breast cancer cells when co-cultured with the stromal fibroblasts and in invasive breast carcinomas as compared with normal breast biopsies. The regulation and function of EPSTI1 are largely unknown. Our studies showed that EPSTI1 expression is upregulated by Kruppel-like factor 8 (KLF8), a transcriptional factor that is highly over-expressed in breast cancer and critical for cell cycle progression, oncogenic transformation, epithelial to mesenchymal transition (EMT) and metastatic progression of breast cancer, by directly activating EPSTI1 promoter. Also, we demonstrated that EPSTI1 was necessary and sufficient to promote breast cancer cell invasiveness by genetically modifying EPSTI1 expression and Matrigel invasion assays. We also found that EPSTI1 could interact with valosin containing protein (VCP) and subsequently activate NF-kB nuclear function. Finally, our mouse mammary fat pad injection and tail vein injection experiments indicated that ectopic overexpression of EPSTI1 in MCF7 cells could enhance the tumor invasion and metastasis in vivo. These findings suggest that EPSTI1 is a novel target of KLF8 and plays a critical role in the regulation of breast cancer invasion and metastasis. Citation Format: Tianshu Li, Heng Lu, Chao Shen, Satadru Lahiri, Melissa Watson, Debarati Mukherjee, Lin Yu, Jihe Zhao. Identification of epithelial stromal interaction 1 as a novel mediator of breast cancer cell invasion and metastasis downstream of Kruppel-like factor 8. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5161. doi:10.1158/1538-7445.AM2013-5161 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 2687: Krüppel-like factor 8 targets matrix metalloproteinase-14 to promote breast cancer invasion and metastasis .

Kruppel-like factor 8 (KLF8) regulates critical gene transcription and invasion events associated with cancer, including epithelial to mesenchymal transition (EMT) and extracellular matrix degradation. Here, we report a novel KLF8-to- matrix metalloproteinase-14 (MMP14) signaling that promotes human breast cancer cell EMT, invasion and metastasis. Cell-surface protein biotinylation and streptavidin pull-down showed that overexpression of KLF8 induced cell-surface accumulation of MMP14 in MCF-10A cells in a FAK-dependent manner, which was correlated with increased MMP14 activity and the ectodomain shedding of E-cadherin. On the other hand, knockdown of KLF8 in MDA-MB-231 cells reduced expression and activity of MMP14 on the cell surface. Promoter reporter assays and chromatin and oligonucleotide precipitations determined that KLF8 activated the human MMP14 gene promoter both directly and indirectly via beta-catenin. Importantly, Matrigel invasion assays showed that MMP14 was required for the KLF8-induced MCF-10A cell invasion in vitro. Experiments using nude mice demonstrated that MMP-14 overexpression downstream of KLF8 in the MDA-MB-231 cells makes a significant contribution to the invasion and angiogenesis in the local primary tumors and lung metastasis. Taken together, this work identified a novel role and mechanisms for MMP14 downstream of KLF8 in the progression of breast cancer metastasis. Citation Format: Heng Lu, Liu Hu, Lin Yu, Xianhui Wang, Chao Shen, Tianshu Li, Debarati Mukherjee, Satadru Lahiri, Melissa Wason, Jihe Zhao. Kruppel-like factor 8 targets matrix metalloproteinase-14 to promote breast cancer invasion and metastasis . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2687. doi:10.1158/1538-7445.AM2013-2687

Abstract 2129: Regulation of DNA repair by Kruppel-like factor 8

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Kruppel-like factor 8 (KLF8) regulates critical gene transcription and cellular events associated with cancer. Recently we have identified poly(ADP-ribose) polymerase-1(PARP-1) as a novel KLF8-interacting and regulating protein. However, whether KLF8 is involved in PARP-1 relulated DNA repair is unknown. Here, we report that KLF8 plays a role in the PARP-1-dependent DNA repair. Western blotting and immunofluorescence staining indicated that overexpression of KLF8 attenuated the αH2A.X levels in doxorubicin-treated PARP-1+/+ MEF cells but not in the PARP-1−/− MEF cells, whereas the PARP-1-interaction-defective KLF8 mutant did not affect the αH2A.X levels. Clonogenic assay and tetrazolium salt (WST-1) assay showed that KLF8 promoted cell survival in human breast cancer cells. Comet assay revealed that KLF8 expression could decrease DNA damage levels. Interestingly, we found that in response to DNA damage KLF8 was phosphorylated by DNA-PKcs and subsequently sumoylated, which depended on the interaction between KLF8 and PARP-1. These results suggest a novel role and mechanism for KLF8 in the regulation of DNA repair in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2129. doi:1538-7445.AM2012-2129