Mehlika Hazar-Rethinam

E2F7 Can Regulate Proliferation, Differentiation, and Apoptotic Responses in Human Keratinocytes: Implications for Cutaneous Squamous Cell Carcinoma Formation

The E2F family of transcription factors plays a crucial role in the regulation of genes involved in cell proliferation, differentiation, and apoptosis. In keratinocytes, the inhibition of E2F is a key step in the control and initiation of squamous differentiation. Because the product of the recently identified E2F7a/E2F7b gene has been shown to repress E2F-regulated promoters, and to be abundant in skin, we examined its role in the epidermis. Our results indicate that E2F7b mRNA expression is selectively associated with proliferation-competent keratinocytes. Moreover, E2F7 was able to antagonize E2F1-induced proliferation and apoptosis. In contrast, although E2F7 was able to inhibit proliferation and initiate differentiation, it was unable to antagonize the differentiation suppression induced by E2F1. These data indicate that E2F7-mediated suppression of proliferation and apoptosis acts through E2F1-dependent pathways, whereas E2F7-induced differentiation acts through an E2F1-independent pathway. These data also suggest that proliferation, differentiation, and survival of primary human keratinocytes can be controlled by the relative ratio of E2F1 to E2F7. Because deregulated proliferation, differentiation, and apoptosis are hallmarks of cancer, we examined the expression levels of E2F1 and E2F7 in cutaneous squamous cell carcinomas (CSCC). We found that both genes were overexpressed in CSCCs compared with normal epidermis. Furthermore, inhibition of E2F7 in a SCC cell line sensitized the cells to UV-induced apoptosis and doxorubicin-induced apoptosis. Combined, these data suggest that the selected disruption of E2F1 and E2F7 in keratinocytes is likely to contribute to CSCC formation and may prove to be a viable therapeutic target.

Preclinical evaluation of dual PI3K-mTOR inhibitors and histone deacetylase inhibitors in head and neck squamous cell carcinoma

Background:We examine the potential value of a series of clinically relevant PI3K-mTOR inhibitors alone, or in combination with histone deacetylase inhibitors, in a model of head and neck squamous cell carcinoma (HNSCC).Methods:Head and neck squamous cell carcinoma cell lines, human keratinocyte and HNSCC xenograft models were treated with histone deacetylase inhibitors (HDACIs) and new generation PI3K and dual PI3K-mTOR inhibitors either alone or in combination. Cell and tumour tissue viability and proliferation were then determined in vitro and in vivo.Results:Phosphatidylinositol-3-phosphate kinase, AKT and dual PI3K-mTOR inhibitors caused marked in vitro enhancement of cytotoxicity induced by HDACIs in HNSCC cancer cells. This effect correlates with AKT inhibition and is attenuated by expression of constitutively active AKT. Histone deacetylase inhibitor and phosphatidylinositol-3-phosphate kinase inhibitors (PI3KIs) inhibited tumour growth in xenograft models of HNSCC. Importantly, we observed intratumoural HDAC inhibition and PI3K inhibition as assessed by histone H3 acetylation status and phospho-AKT staining, respectively. However, we saw no evidence of improved efficacy with an HDACI/PI3KI combination.Interpretation:That PI3K and dual PI3K-mTOR inhibitors possess antitumour effect against HNSCC in vivo.

Resistance to checkpoint blockade therapy through inactivation of antigen presentation

Treatment with immune checkpoint blockade (CPB) therapies often leads to prolonged responses in patients with metastatic melanoma, but the common mechanisms of primary and acquired resistance to these agents remain incompletely characterized and have yet to be validated in large cohorts. By analyzing longitudinal tumor biopsies from 17 metastatic melanoma patients treated with CPB therapies, we observed point mutations, deletions or loss of heterozygosity (LOH) in beta-2-microglobulin (B2M), an essential component of MHC class I antigen presentation, in 29.4% of patients with progressing disease. In two independent cohorts of melanoma patients treated with anti-CTLA4 and anti-PD1, respectively, we find that B2M LOH is enriched threefold in non-responders (~30%) compared to responders (~10%) and associated with poorer overall survival. Loss of both copies of B2M is found only in non-responders. B2M loss is likely a common mechanism of resistance to therapies targeting CTLA4 or PD1.Resistance to immune-checkpoint blockade often occurs in treated patients. Here, the authors demonstrate that B2M loss is a mechanism of primary and acquired resistance to therapies targeting CTLA4 or PD-1 in melanoma patients.

Dysregulation of the Repressive H3K27 Trimethylation Mark in Head and Neck Squamous Cell Carcinoma Contributes to Dysregulated Squamous Differentiation

Purpose: Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent cancers diagnosed worldwide and is associated with a 5-year survival rate of 55%. EZH2, a component of the polycomb repressor complex 2, trimethylates H3K27 (H3K27me3), which has been shown to drive squamous differentiation in normal keratinocytes. This study determined whether inhibition of EZH2-mediated epigenetic silencing could induce differentiation or provide therapeutic benefit in HNSCC. Experimental Design: We determined the effects of inhibiting EZH2, by either RNA interference or pharmacologically, on HNSCC growth, viability, and differentiation in vitro. Xenografts of HNSCC cell lines were used to assess efficacy of 3-deazaneplanocin A (DZNep), an inhibitor of H3K27 trimethylation, in vivo. Results: EZH2 was highly expressed in HNSCC cell lines in vitro and tissue microarray analysis revealed high expression in (n = 59) in situ relative to normal oral epithelium (n = 12). Inhibition of EZH2 with siRNA could induce expression of differentiation genes in differentiation-refractory squamous cell carcinoma cell lines. Differentiation-refractory HNSCC cell lines displayed persistent H3K27me3 on the promoters of differentiation genes. DZNep caused cancer-cell–specific apoptosis in addition to a profound reduction in colony-forming efficiency and induction of some squamous differentiation genes. Furthermore, in vivo, DZNep attenuated tumor growth in two different xenograft models, caused intratumor inhibition of EZH2, and induction of differentiation genes in situ. Conclusions: Collectively, these data suggest that aberrant differentiation in HNSCC may be attributed to epigenetic dysregulation and suggest that inhibition of PRC2-mediated gene repression may represent a potential therapeutic target. Clin Cancer Res; 19(2); 428–41. ©2012 AACR.

A Novel E2F/Sphingosine Kinase 1 Axis Regulates Anthracycline Response in Squamous Cell Carcinoma

Purpose: Head and neck squamous cell carcinomas (HNSCC) are frequently drug resistant and have a mortality rate of 45%. We have previously shown that E2F7 may contribute to drug resistance in SCC cells. However, the mechanism and pathways involved remain unknown. Experimental Design: We used transcriptomic profiling to identify candidate pathways that may contribute to E2F7-dependent resistance to anthracyclines. We then manipulated the activity/expression of the candidate pathway using overexpression, knockdown, and pharmacological inhibitors in in vitro and in vivo models of SCC to demonstrate causality. In addition, we examined the expression of E2F7 and a downstream effector in a tissue microarray (TMA) generated from HNSCC patient samples. Results: E2F7-deficient keratinocytes were selectively sensitive to doxorubicin and this was reversed by overexpressing E2F7. Transcriptomic profiling identified Sphingosine kinase 1 (Sphk1) as a potential mediator of E2F7-dependent drug resistance. Knockdown and overexpression studies revealed that Sphk1 was a downstream target of E2F7. TMA studies showed that E2F7 overexpression correlated with Sphk1 overexpression in human HNSCC. Moreover, inhibition of Sphk1 by shRNA or the Sphk1-specific inhibitor, SK1-I (BML-EI411), enhanced the sensitivity of SCC cells to doxorubicin in vitro and in vivo. Furthermore, E2F7-induced doxorubicin resistance was mediated via Sphk1-dependent activation of AKT in vitro and in vivo. Conclusion: We identify a novel drugable pathway in which E2F7 directly increases the transcription and activity of the Sphk1/S1P axis resulting in activation of AKT and subsequent drug resistance. Collectively, this novel combinatorial therapy can potentially be trialed in humans using existing agents. Clin Cancer Res; 21(2); 417–27. ©2014 AACR.

The Role of the E2F Transcription Factor Family in UV-Induced Apoptosis

The E2F transcription factor family is traditionally associated with cell cycle control. However, recent data has shown that activating E2Fs (E2F1-3a) are potent activators of apoptosis. In contrast, the recently cloned inhibitory E2Fs (E2F7 and 8) appear to antagonize E2F-induced cell death. In this review we will discuss (i) the potential role of E2Fs in UV-induced cell death and (ii) the implications of this to the development of UV-induced cutaneous malignancies.

Focal overexpression of CEACAM6 contributes to enhanced tumourigenesis in head and neck cancer via suppression of apoptosis

BackgroundOverexpression of CEACAM6 has been reported for a number of malignancies. However, the mechanism of how CEACAM6 contributes to cancer formation and its role in head and neck squamous cell carcinoma (HNSCC) remains unclear. Therefore, we examined the role of CEACAM6 in head and neck squamous cell carcinoma (HNSCC).MethodsCEACAM6 expression was examined in normal squamous epithelia as well as a number of patient HNSCC samples and tumours derived from HNSCC cell lines injected into NOD/SCID mice. CEACAM6 expression was manipulated in HNSCC cell lines by shRNA-mediated CEACAM6 knockdown or virally-delivered overexpression of CEACAM6. The role of CEACAM6 in tumour growth and chemotherapeutic sensitivity was then assessed in vivo and in vitro respectively.ResultsCEACAM6 expression was significantly increased in highly tumourigenic HNSCC cell lines when compared to poorly tumourigenic HNSCC cell lines. Moreover, HNSCC patient tumours demonstrated focal expression of CEACAM6. Functional investigation of CEACAM6, involving over-expression and knock down studies, demonstrated that CEACAM6 over-expression could enhance tumour initiating activity and tumour growth via activation of AKT and suppression of caspase-3 mediated cell death.ConclusionWe report that CEACAM6 is focally overexpressed in a large fraction of human HNSCCs in situ. We also show that over-expression of CEACAM6 increases tumour growth and tumour initiating activity by suppressing PI3K/AKT-dependent apoptosis of HNSCC in a xenotransplant model of HNSCC. Finally, our studies indicate that foci of CEACAM6 expressing cells are selectively ablated by treatment of xenotransplant tumours with pharmacological inhibitors of PI3K/AKT in vivo.

Loss of E2F7 expression is an early event in squamous differentiation and causes derepression of the key differentiation activator Sp1.

Squamous differentiation is controlled by key transcription factors such as Sp1 and E2F. We have previously shown that E2F1 can suppress transcription of the differentiation-specific gene, transglutaminase type 1 (TG1), by an indirect mechanism mediated by Sp1. Transient transfection of E2F1-E2F6 indicated that E2F-mediated reduction of Sp1 transcription was not responsible for E2F-mediated suppression of squamous differentiation. However, we found that E2F4 and E2F7, but not E2Fs 1, 2, 3, 5, or 6, could suppress the activation of the Sp1 promoter in differentiated keratinocytes (KCs). E2F4-mediated suppression could not be antagonized by E2Fs 1, 2, 3, 5, or 6 and was localized to a region of the human Sp1 promoter spanning -139 to + 35 bp. Chromatin immunoprecipitation analysis, as well as transient overexpression and short hairpin RNA knockdown experiments indicate that E2F7 binds to a unique binding site located between -139 and -119 bp of the Sp1 promoter, and knockdown of E2F7 in proliferating KCs leads to a derepression of Sp1 expression and the induction of TG1. In contrast, E2F4 knockdown in proliferating KCs did not alter Sp1 expression. These data indicate that loss of E2F7 during the initiation of differentiation leads to the derepression of Sp1 and subsequent transcription of differentiation-specific genes such as TG1.

Auranofin is a potent suppressor of osteosarcoma metastasis

Osteosarcoma (OS) accounts for 56% of malignant bone cancers in children and adolescents. Patients with localized disease rarely develop metastasis; however, pulmonary metastasis occurs in approximately 50% of patients and leads to a 5-year survival rate of only 10–20%. Therefore, identifying the genes and pathways involved in metastasis, as new therapeutic targets, is crucial to improve long-term survival of OS patients. Novel markers that define metastatic OS were identified using comparative transcriptomic analyses of two highly metastatic (C1 and C6) and two poorly metastatic clonal variants (C4 and C5) isolated from the metastatic OS cell line, KHOS. Using this approach, we determined that the metastatic phenotype correlated with overexpression of thioredoxin reductase 2 (TXNRD2) or vascular endothelial growth factor (VEGF). Validation in patient biopsies confirmed TXNRD2 and VEGF targets were highly expressed in 29–42% of metastatic OS patient biopsies, with no detectable expression in non-malignant bone or samples from OS patients with localised disease. Auranofin (AF) was used to selectively target and inhibit thioredoxin reductase (TrxR). At low doses, AF was able to inhibit TrxR activity without a significant effect on cell viability whereas at higher doses, AF could induce ROS-dependent apoptosis. AF treatment, in vivo, significantly reduced the development of pulmonary metastasis and we provide evidence that this effect may be due to an AF-dependent increase in cellular ROS. Thus, TXNRD2 may represent a novel druggable target that could be deployed to reduce the development of fatal pulmonary metastases in patients with OS.

Real-time Genomic Characterization of Advanced Pancreatic Cancer to Enable Precision Medicine

Clinically relevant subtypes exist for pancreatic ductal adenocarcinoma (PDAC), but molecular characterization is not yet standard in clinical care. We implemented a biopsy protocol to perform time-sensitive whole-exome sequencing and RNA sequencing for patients with advanced PDAC. Therapeutically relevant genomic alterations were identified in 48% (34/71) and pathogenic/likely pathogenic germline alterations in 18% (13/71) of patients. Overall, 30% (21/71) of enrolled patients experienced a change in clinical management as a result of genomic data. Twenty-six patients had germline and/or somatic alterations in DNA-damage repair genes, and 5 additional patients had mutational signatures of homologous recombination deficiency but no identified causal genomic alteration. Two patients had oncogenic in-frame BRAF deletions, and we report the first clinical evidence that this alteration confers sensitivity to MAPK pathway inhibition. Moreover, we identified tumor/stroma gene expression signatures with clinical relevance. Collectively, these data demonstrate the feasibility and value of real-time genomic characterization of advanced PDAC.Significance: Molecular analyses of metastatic PDAC tumors are challenging due to the heterogeneous cellular composition of biopsy specimens and rapid progression of the disease. Using an integrated multidisciplinary biopsy program, we demonstrate that real-time genomic characterization of advanced PDAC can identify clinically relevant alterations that inform management of this difficult disease. Cancer Discov; 8(9); 1096-111. ©2018 AACR.See related commentary by Collisson, p. 1062This article is highlighted in the In This Issue feature, p. 1047.