Yuhong Lu

Molecular mechanisms underlying IGF‐I‐induced attenuation of the growth‐inhibitory activity of trastuzumab (Herceptin) on SKBR3 breast cancer cells

The clinical usefulness of trastuzumab (Herceptin; Genentech, San Francisco, CA) in breast cancer treatment is limited by the rapid development of resistance. We previously reported that IGF‐I signaling confers resistance to the growth‐inhibitory actions of trastuzumab in a model system, but the underlying molecular mechanism remains unknown. We used SKBR3/neo cells (expressing few IGF‐I receptors) and SKBR3/IGF‐IR cells (overexpressing IGF‐I receptor) as our experimental model. IGF‐I antagonized the trastuzumab‐induced increase in the level of the Cdk inhibitor p27Kip1. This resulted in decreased association of p27Kip1 with Cdk2, restoration of Cdk2 activity and attenuation of cell‐cycle arrest in G1 phase, all of which had been induced by trastuzumab treatment in SKBR3/IGF‐IR cells. We also found that the decrease in p27Kip1 induced by IGF‐I was accompanied by an increase in expression of Skp2, which is a ubiquitin ligase for p27Kip1, and by increased Skp2 association with p27Kip1. A specific proteasome inhibitor (LLnL) completely blocked the ability of IGF‐I to reduce the p27Kip1 protein level, while IGF‐I increased p27Kip1 ubiquitination. This suggests that the action of IGF‐I in conferring resistance to trastuzumab involves targeting of p27Kip1 to the ubiquitin/proteasome degradation machinery. Finally, specific inhibitors of MAPK and PI3K suggest that the IGF‐I‐mediated reduction in p27Kip1 protein level by increased degradation predominantly involves the PI3K pathway. Our results provide an example of resistance to an antineoplastic therapy that targets one tyrosine kinase receptor by increased signal transduction through an alternative pathway in a complex regulatory network.

Inhibition of poly(ADP-ribose) polymerase down-regulates BRCA1 and RAD51 in a pathway mediated by E2F4 and p130

Inhibitors of poly(ADP-ribose) polymerase (PARP) are in clinical trials for cancer therapy, on the basis of the role of PARP in recruitment of base excision repair (BER) factors to sites of DNA damage. Here we show that PARP inhibition to block BER is toxic to hypoxic cancer cells, in which homology-dependent repair (HDR) is known to be down-regulated. However, we also report the unexpected finding that disruption of PARP, itself, either via chemical PARP inhibitors or siRNAs targeted to PARP-1, can inhibit HDR by suppressing expression of BRCA1 and RAD51, key factors in HDR of DNA breaks. Mechanistically, PARP inhibition was found to cause increased occupancy of the BRCA1 and RAD51 promoters by repressive E2F4/p130 complexes, a pathway prevented by expression of HPV E7, which disrupts p130 activity, or by siRNAs to knock down p130 expression. Functionally, disruption of p130 by E7 expression or by siRNA knockdown also reverses the cytotoxicity and radiosensitivity associated with PARP inhibition, suggesting that the down-regulation of BRCA1 and RAD51 is central to these effects. Direct measurement of HDR using a GFP-based assay demonstrates reduced HDR in cells treated with PARP inhibitors. This work identifies a mechanism by which PARP regulates DNA repair and suggests new strategies for combination cancer therapies.

Hypoxia-Induced Epigenetic Regulation and Silencing of the BRCA1 Promoter

ABSTRACT Disruption of the BRCA1 tumor suppressor can be caused not only by inherited mutations in familial cancers but also by BRCA1 gene silencing in sporadic cancers. Hypoxia, a key feature of the tumor microenvironment, has been shown to downregulate BRCA1 at the transcriptional level via repressive E2F4/p130 complexes. Here we showed that hypoxia also drives epigenetic modification of the BRCA1 promoter, with decreased H3K4 methylation as a key repressive modification produced by the lysine-specific histone demethylase LSD1. We also observed increased H3K9 methylation coupled with decreased H3K9 acetylation. Similar modifications were seen in the RAD51 promoter, which is also downregulated by hypoxia, whereas exactly opposite changes were seen in the promoter of the hypoxia-inducible gene VEGF. In cells containing the BRCA1 promoter driving a selectable HPRT gene, long-term silencing of the promoter was observed following exposure to hypoxic stress. Clones with silenced BRCA1 promoters were detected at frequencies of 2% or more following hypoxia, but at less than 6 × 10−5 without hypoxia. The silenced clones showed decreased H3K4 methylation and decreased H3K9 acetylation in the BRCA1 promoters, consistent with the acute effects of hypoxic stress. Hypoxia-induced BRCA1 promoter silencing persisted in subsequent normoxic conditions but could be reversed by treatment with a histone deacetylase (HDAC) inhibitor but not with a DNA methylation inhibitor. Interestingly, treatment of cells with inhibitors of poly(ADP-ribose) polymerase (PARP) can cause short-term repression of BRCA1 expression, but such treatment does not produce H3K4 or H3K9 histone modification or BRCA1 promoter silencing. These results suggest that hypoxia is a driving force for long-term silencing of BRCA1, thereby promoting genome instability and tumor progression.

Hypoxia Promotes Resistance to EGFR Inhibition in NSCLC Cells via the Histone Demethylases, LSD1 and PLU-1

The development of small-molecule tyrosine kinase inhibitors (TKI) specific for epidermal growth factor receptors (EGFR) with activating mutations has led to a new paradigm in the treatment of non–small cell lung cancer (NSCLC) patients. However, most patients eventually develop resistance. Hypoxia is a key microenvironmental stress in solid tumors that is associated with poor prognosis due, in part, to acquired resistance to conventional therapy. This study documents that long-term, moderate hypoxia promotes resistance to the EGFR TKI, gefitinib, in the NSCLC cell line HCC827, which harbors an activating EGFR mutation. Following hypoxic growth conditions, HCC827 cells treated with gefitinib upregulated N-cadherin, fibronectin, and vimentin expression and downregulated E-cadherin, characteristic of an epithelial-mesenchymal transition (EMT), which prior studies have linked to EGFR TKI resistance. Mechanistically, knockdown of the histone demethylases, LSD1 and PLU-1, prevented and reversed hypoxia-induced gefitinib resistance, with inhibition of the associated EMT, suggesting that LSD1 and PLU-1 play key roles in hypoxia-induced gefitinib resistance and EMT. Moreover, hypoxia-treated HCC827 cells demonstrated more aggressive tumor growth in vivo compared with cells grown in normoxia, but inhibition of LSD1 function by shRNA-mediated knockdown or by the small-molecular inhibitor SP2509 suppressed tumor growth and enhanced gefitinib response in vivo. These results suggest that hypoxia is a driving force for acquired resistance to EGFR TKIs through epigenetic change and coordination of EMT in NSCLC. This study suggests that combination of therapy with EGFR TKIs and LSD1 inhibitors may offer an attractive therapeutic strategy for NSCLCs. Mol Cancer Res; 16(10); 1458–69. ©2018 AACR.

Abstract 736: Hypoxia-induced EGFR tyrosine kinase inhibitor resistance is associated with epithelial-mesenchymal transition in NSCLC

The development of small molecule inhibitors specific for epidermal growth factor receptors (EGFR) with activating mutations has led to a dramatic shift in the treatment of non-small cell lung cancer (NSCLC) patients. Patients with NSCLC carrying such EGFR mutations often show prolonged responses when treated with first or second generation EGFR tyrosine kinase inhibitors (TKIs). However, most patients eventually develop resistance to EGFR TKIs. Hypoxia is a key micro-environmental stress in solid tumors that is associated with poor prognosis. Studies have linked hypoxia to therapy resistance, including radiation therapy and chemotherapy. In this study, we show that long-term, moderate hypoxia induces gefitinib resistance in the NSCLC cell line, HCC827, that harbors an activating EGFR mutation. The hypoxia-induced gefitinib resistance is associated with reduced BIM induction after gefitinib treatment when compared to normoxic HCC827 cells. In addition, in hypoxic HCC827 cells, gefitinib treatment induces N-cadherin expression, a mesenchymal marker, accompanied by downregulation of E-cadherin, an epithelial maker. These results suggest that epithelial-mesenchymal transition (EMT) may be involved in hypoxia induced gefitinib resistance. Wound healing migration assays further support that hypoxia induces HCC827 cell migration after gefitinib treatment. These results suggest that hypoxia may be a driving force for EGFR TKIs resistance potentially through EMT in NSCLC. Citation Format: Yuhong Lu, Peter M. Glazer. Hypoxia-induced EGFR tyrosine kinase inhibitor resistance is associated with epithelial-mesenchymal transition in NSCLC. [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 736.

Abstract 1513: MLH1 silencing is caused by hypoxia through epigenetic regulation..

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Transcriptional silencing of the DNA mismatch repair (MMR) gene, MLH1, is one of the main causes of microsatellite instability in sporadic cancers of the colon and other sites. We and others have shown that hypoxia, a key microenvironmental stress in solid tumors, causes reduced expression of MLH1via transient transcriptional repression mediated by both HIF-dependent and independent pathways. Here, we further investigated whether hypoxia-induced down-regulation of MLH1 is associated with epigenetic modifications that may mark the locus for long-term silencing. We find that hypoxia drives epigenetic repression of the MLH1 promoter with decreased H3K4 methylation as a key repressive modification. We find that this is mediated by the H3K4 demethylases, LSD1 (lysine-specific histone demethylase 1) and PLU-1/JARID1B. In RKO cells containing a reporter construct consisting of the MLH1 promoter driving selectable thymidine kinase (TK) and blasticidin resistance (BlastR) genes, long-term silencing of the MLH1 promoter was produced by exposure of the cells to hypoxia as compared to minimal silencing under normoxia. Importantly, knockdown of LSD1 blocked the hypoxia-induced MLH1 promoter silencing in this reporter system. In RKO cells, the endogenous MLH1 promoter is constitutively silenced by DNA hypermethylation at CpG sites. MLH1 re-expression can be induced by 5-aza-2-deoxycytidine (5-Aza-dC), a DNA methyltransferase inhibitor, accompanied by increased levels of H3K9 acetylation and H3K4 methylation at it promoter. But the promoter rapidly becomes re-silenced after removal of 5-Aza-dC. However, we find that knockdown of LSD1 or its corepressor, CoRest, blocks MLH1 resilencing in the RKO cells. Consistently, ChIP analyses revealed that the control RKO cells (with a GFP knockdown vector) showed return of both H3K9 acetylation and H3K4 methylation back to pre-treatment levels coupled with MLH1 re-silencing upon removal of 5-Aza-dC, but intriguingly, high levels of H3K9 acetylation and H3K4 methylation were also observed at MLH1 promoters in RKO LSD1sh cells. Analysis of DNA methylation revealed that MLH1 promoter methylation levels were reduced by 5-Aza-dC treatment, as expected. However, after removal of 5-Aza-dC, promoter methylation levels returned back to the pre-treatment levels only in the control RKO cells. In the RKO cells with shRNA mediated knock down of either LSD1 or CoRest, the promoter re-methylation process was blocked. Collectively, the results demonstrate that the LSD1/CoRest repressive complex is essential for the MLH1 silencing and that hypoxia is a critical driving force for stable transcriptional silencing of MLH1 through a concerted pattern of chromatin and DNA modifications, thereby promoting genetic instability and tumor progression. Citation Format: Yuhong Lu, Narendra Wajapeyee, Mitchell S. Turker, Peter M. Glazer. MLH1 silencing is caused by hypoxia through epigenetic regulation. [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 1513. doi:10.1158/1538-7445.AM2013-1513

Abstract 3939: Hypoxia-induced BRCA1 phosphorylation and degradation

Hypoxia is a key feature of tumor microenviroment. We have demonstrated that hypoxia downregulates BRCA1 at the transcriptional level through promoter binding by E2F4/p130 complexes and that hypoxia can produce BRCA1 gene silencing via epigenetic modifications. We also demonstrated that downregulation of BRCA1 by hypoxia leads to decreased DNA repair capacity, establishing a mechanism by which hypoxia can drive genetic instability in cancer cells. In addition to transcriptional regulation, BRCA1 has been found to be phosphorylated at S988 in a Chk2-dependent manner by both ionizing radiation and hypoxia exposure. In this study, we have been able to confirm that other residues of BRCA1 are phosphoylated during hypoxia exposure, including S1387, S1423, S1466, and S1524. Intriguingly, the phosphoryation of BRCA1 only happens under severe hypoxic condition, such as 0.01% oxygen, but not under moderate hypoxia (1% oxygen condition). Furthermore, the BRCA1 phosphoylation events induced by hypoxia can be attenuated by an ATM inhibitor, KU55933, indicating that ATM is playing a role in hypoxia-induced BRCA1 phosphorylation at multiple sites. We also observed that the decrease in BRCA1 protein levels in hypoxia is partially blocked by MG132, a proteasome inhibitor. Consistent with this, by transient transfection of exogenous BRCA1, we found that hypoxia induces BRCA1 unbiquitination in MCF-7 cells. Together, our data suggest that hypoxia-induced downregulation of BRCA1 is in part mediated through the ubiqutin-proteasome pathway. Our findings indicate a new mechanism of hypoxia induced regulation of BRCA1 by modulating its stability, suggesting that hypoxia-induced downregulation of BRCA1 is through multiple mechanisms. Future studies will investigate the mechanism of hypoxia-induced BRCA1 phophorylation and the relation between hypoxia-induced BRCA1 phosphorylation and BRCA1 degradation. To understand more about regulation of BRCA1 by hypoxia could lead to new therapies to target hypoxic tumors. 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 3939. doi:1538-7445.AM2012-3939

Abstract 1969: Inhibition of poly(ADP-ribose) polymerase downregulates BRCA1 and RAD51 in a pathway mediated by E2F4 and p130

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Inhibitors of poly(ADP-ribose) polymerase (PARP) are in clinical trials for cancer therapy based on the role of PARP in recruitment of base excision repair (BER) factors to sites of damage. Here we show that PARP inhibition to block BER is toxic to hypoxic cancer cells, in which homology-dependent repair (HDR) is known to be down-regulated. However, we also report the unexpected finding that disruption of PARP, itself, either via chemical PARP inhibitors or siRNAs targeted to PARP-1, can inhibit HDR by suppressing expression of BRCA1 and RAD51, key factors in HDR of DNA breaks. Mechanistically, PARP inhibition was found to cause increased occupancy of the BRCA1 and RAD51 promoters by repressive E2F4/p130 complexes, a pathway prevented by expression of HPV E7, which disrupts p130 activity, or by siRNAs to knock down p130 expression. Functionally, disruption of p130 by E7 expression or by siRNA knockdown also reverses the cytotoxicity and radiosensitivity associated with PARP inhibition, suggesting that the down-regulation of BRCA1 and RAD51 is central to these effects. Direct measurement of HDR using a GFP-based assay demonstrates reduced HDR in cells treated with PARP inhibitors. This work identifies a novel mechanism by which PARP regulates DNA repair and suggests new strategies for combination cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1969.