Vida Chitsazzadeh

Cross-species identification of genomic drivers of squamous cell carcinoma development across preneoplastic intermediates

Cutaneous squamous cell carcinoma (cuSCC) comprises 15–20% of all skin cancers, accounting for over 700,000 cases in USA annually. Most cuSCC arise in association with a distinct precancerous lesion, the actinic keratosis (AK). To identify potential targets for molecularly targeted chemoprevention, here we perform integrated cross-species genomic analysis of cuSCC development through the preneoplastic AK stage using matched human samples and a solar ultraviolet radiation-driven Hairless mouse model. We identify the major transcriptional drivers of this progression sequence, showing that the key genomic changes in cuSCC development occur in the normal skin to AK transition. Our data validate the use of this ultraviolet radiation-driven mouse cuSCC model for cross-species analysis and demonstrate that cuSCC bears deep molecular similarities to multiple carcinogen-driven SCCs from diverse sites, suggesting that cuSCC may serve as an effective, accessible model for multiple SCC types and that common treatment and prevention strategies may be feasible.

Sorafenib Suppresses JNK-Dependent Apoptosis through Inhibition of ZAK

Sorafenib is U.S. Food and Drug Adminstration–approved for the treatment of renal cell carcinoma and hepatocellular carcinoma and has been combined with numerous other targeted therapies and chemotherapies in the treatment of many cancers. Unfortunately, as with other RAF inhibitors, patients treated with sorafenib have a 5% to 10% rate of developing cutaneous squamous cell carcinoma (cSCC)/keratoacanthomas. Paradoxical activation of extracellular signal–regulated kinase (ERK) in BRAF wild-type cells has been implicated in RAF inhibitor–induced cSCC. Here, we report that sorafenib suppresses UV-induced apoptosis specifically by inhibiting c-jun–NH2–kinase (JNK) activation through the off-target inhibition of leucine zipper and sterile alpha motif–containing kinase (ZAK). Our results implicate suppression of JNK signaling, independent of the ERK pathway, as an additional mechanism of adverse effects of sorafenib. This has broad implications for combination therapies using sorafenib with other modalities that induce apoptosis. Mol Cancer Ther; 13(1); 221–9. ©2013 AACR.

Differential T-cell subset representation in cutaneous squamous cell carcinoma arising in immunosuppressed versus immunocompetent individuals.

carcinoma arising in immunosuppressed versus immunocompetent individuals Laurence Feldmeyer, Grace Ching, Harina Vin, Wencai Ma, Varun Bansal, Vida Chitsazzadeh, Richard Jahan-Tigh, Emily Y. Chu, Peter Fuller, Sourindra Maiti, Richard Eric Davis, Laurence J.N. Cooper and Kenneth Y. Tsai Department of Translational Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Pathology, University of Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Lymphoma & Myeloma, University of Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Dermatology, University of Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Dermatology, University of Pennsylvania, Philadelphia, PA, USA; Division of Pediatrics, University of Texas MD Anderson Cancer Center, Houston, TX, USA Correspondence: Kenneth Y. Tsai, MD, PhD, Departments of Dermatology & Translational Molecular Pathology, University of Texas MD Anderson Cancer Center, Unit 2951, 2130 West Holcombe Blvd., LSP 9.4218, Houston, TX 77030, USA, Tel.: +1 713-563-1943, e-mail: kytsai@mdanderson.org

Deficient synthesis of class-switched, Hiv-neutralizing antibodies to the Cd4 binding site and correction by electrophilic gp120 immunogen

Objective:HIV is vulnerable to antibodies that recognize a linear CD4 binding site epitope of gp120 (CLIN), but inducing CLIN-directed antibody synthesis by traditional vaccine principles is difficult. We wished to understand the basis for deficient CLIN-directed antibody synthesis and validate correction of the deficiency by an electrophilic gp120 analog (E-gp120) immunogen that binds B-cell receptors covalently. Methods:Serum antibody responses to a CLIN peptide and full-length gp120 epitopes induced by HIV infection in humans and immunization of mice with gp120 or E-gp120 were monitored. HIV neutralization by monoclonal and variable domain-swapped antibodies was determined from tissue culture and humanized mouse infection assays. Results:We describe deficient CLIN-directed IgG but not IgM antibodies in HIV-infected patients and mice immunized with gp120 accompanied by robust synthesis of IgGs to the immunodominant gp120 epitopes. Immunization with the E-gp120 corrected the deficient CLIN-directed IgG synthesis without overall increased immunogenicity of the CLIN or other gp120 epitopes. E-gp120-induced monoclonal IgGs neutralized diverse HIV strains heterologous to the immunogen. A CLIN-directed IgG neutralized HIV more potently compared to its larger IgM counterpart containing the same variable domains, suggesting obstructed access to HIV surface-expressed CLIN. An E-gp120-induced IgG suppressed HIV infection in humanized mice, validating the tissue culture neutralizing activity. Conclusion:A CLIN-selective physiological defect of IgM→IgG class-switch recombination (CSR) or restricted post-CSR B-cell development limits the functional utility of the humoral immune response to gp120. The E-gp120 immunogen is useful to bypass the restriction and induce broadly neutralizing CLIN-directed IgGs (see Supplemental Video Abstract, http://links.lww.com/QAD/A551).

TCF7L1 promotes skin tumorigenesis independently of β-catenin through induction of LCN2

The transcription factor TCF7L1 is an embryonic stem cell signature gene that is upregulated in multiple aggressive cancer types, but its role in skin tumorigenesis has not yet been defined. Here we document TCF7L1 upregulation in skin squamous cell carcinoma (SCC) and demonstrate that TCF7L1 overexpression increases tumor incidence, tumor multiplicity, and malignant progression in the chemically induced mouse model of skin SCC. Additionally, we show that downregulation of TCF7L1 and its paralogue TCF7L2 reduces tumor growth in a xenograft model of human skin SCC. Using separation-of-function mutants, we show that TCF7L1 promotes tumor growth, enhances cell migration, and overrides oncogenic RAS-induced senescence independently of its interaction with β-catenin. Through transcriptome profiling and combined gain- and loss-of-function studies, we identified LCN2 as a major downstream effector of TCF7L1 that drives tumor growth. Our findings establish a tumor-promoting role for TCF7L1 in skin and elucidate the mechanisms underlying its tumorigenic capacity. DOI: http://dx.doi.org/10.7554/eLife.23242.001

Abstract 43: MicroRNA-130b mediates a metabolic switch to promote cutaneous squamous cell carcinoma development

Metabolic reprogramming has been emerging as a hallmark of cancer. Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer, with about 700,000 new cases annually in the U.S. We aim to investigate the role of miR-130b in reprogramming metabolism as a driver of cSCC development. Since skin cancers have the highest mutational loads among any human cancers, we used well-controlled comparisons across tissues and across species to narrow down the number of important candidate drivers. Using the approach of matched isogenic human samples and cross-species analysis using our UV-driven hairless mouse model, we identified aberrantly expressed microRNAs (miRNAs) and their target mRNAs in order to unravel additional mechanisms behind cSCC progression. We focus on miRNAs because they regulate numerous mRNA targets and can be manipulated for cancer therapy. We observed that miR-130b is increased in both human and mouse cSCC by 3.3-fold, and 2.6-fold, respectively. In adipocytes, miR-130b is known to reduce fat deposition and cell differentiation through targeting PPARγ. Our data and the TargetScan algorithm suggested that in keratinocytes, miR-130b suppresses PPARγ, FBP1, PGC-1α and PDK4. These genes are down-regulated in SCC tumors (compared to normal skin). Also, they are regulators of glycolysis, mitochondrial activity and lipid biosynthesis. Specifically, PPARγ promotes lipid biosynthesis; PGC-1α and PDK4 control mitochondrial activity; FBP1 suppresses glycolysis. Thus, we hypothesize that the cSCC metabolic phenotype is driven mostly by glycolysis and that miR-130b is a regulator of this metabolic switch. We found that in cSCC cell lines, proliferation is reduced by up to 30% when miR-130b was inhibited. Also in the same setting, less glucose was consumed and less lactate was produced, suggesting that miR-130b promotes glycolysis. Realtime-PCR shows that miR-130b levels are up-regulated in SCC tumors and in SCC cell lines (compared to normal skin and primary keratinocytes, respectively). On the other hand, PPARγ and PGC-1α gene expression decreases during SCC development. To study the long-term effects of miR-130b depletion, we have also developed an inducible CRISPRi system that suppresses microRNA expression. Finally, rosiglitazone, a PPARγ agonist, suppresses the proliferation of several SCC cell lines and is under several trials for breast cancer and liposarcoma, thus suggesting that this pathway may be targetable in SCC as well. Our current studies are focused on the precise roles of PPARγ transcriptional targets, which may include FBH1, PGC-1α and PDK4 in cSCC. Citation Format: Tran N. Nguyen, Sydney Moyer, Charles H. Adelmann, Vida Chitsazzadeh, Kenneth Y. Tsai. MicroRNA-130b mediates a metabolic switch to promote cutaneous squamous cell carcinoma development. [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 43.

Abstract 1920: Novel molecular targets for chemoprevention of squamous cell carcinoma

INTRODUCTION: It is estimated that cancer prevention efforts can reduce cancer incidence by over 50%. Unlike for advanced disease, effective molecularly-driven interventions and risk assessment are not available for clinically normal tissues that may be have been exposed to carcinogens or for precancerous lesions. Cutaneous squamous cell carcinoma (cuSCC) comprises 15-20% of all skin cancers and has the most accessible and clinically well-characterized progression sequence of any human cancer, from a distinct precancerous lesion, the actinic keratosis (AK), to invasive carcinoma. Thus, it is an ideal model for establishing a paradigm of molecularly targeted cancer chemoprevention. METHODS: Here, we performed next-generation sequencing of total RNA and miRNA (Illumina HiSeq) on matched isogenic samples of human cuSCC, surrounding normal (chronically irradiated) skin (NS), and AK. In parallel, we profiled matched samples from a UV-driven Hairless mouse model of cuSCC for cross-species analysis, to identify the most important drivers of progression from NS to AK to cuSCC. RESULTS: Unsupervised clustering of both mRNA and miRNA expression changes showed that preneoplastic AKs span a continuum indistinguishable from cuSCC or surrounding NS, whereas cuSCC and NS were easily distinguished. Through cross-species computational analysis of mRNA-miRNA functional pairs, we identified miR-21, miR-205, miR-31, let-7B, and miR-497 and their mRNA targets as core drivers of cuSCC development through the AK intermediate. We show that several of these miRNAs are modulated by UV exposure, regulate susceptibility to apoptosis, and regulate cell motility. Several miRNAs are being inhibited in our mouse model to validate them as chemoprevention targets. TRANSFAC analysis identified E2F, SP1, AP1, and TCF3 as key transcriptional regulators of SCC development. We tested the global mRNA and miRNA expression similarities to other tumor types profiled by the NIH TCGA effort, showing that cuSCC is most closely related to head & neck SCC, lung SCC, and basal subtype of breast cancer. Exome analysis confirmed a high frequency of TP53, NOTCH1/2, and CDKN2A mutations in cuSCC, with mutational loads of >30/Mb that are strongly dominated by UVB signature lesions. Surprisingly, NS contains evidence of numerous mutations reflecting UV-induced somatic mosaicism. CONCLUSIONS: We report the first integrated transcriptomic characterization of the development of cuSCC through the preneoplastic AK. AKs are indistinguishable from cuSCC, suggesting that chemoprevention efforts should be directed at UV-exposed skin prior to the emergence of lesions. Key transcriptional responses in miRNAs and transcription factor networks were identified in our cross-species analysis, which have been functionally validated. Finally, SCCs of diverse anatomic sites share deep genomic commonalities suggesting that there may be common chemoprevention strategies applicable to multiple SCC types. Citation Format: Vida Chitsazzadeh, Cristian Coarfa, Tri H. Nguyen, Aaron K. Joseph, Preethi Gunaratne, Li Shen, Hui Yao, Weimin Xiao, Xiaoping Su, Jennifer Drummond, David Wheeler, Elsa R. Flores, Kenneth Y. Tsai. Novel molecular targets for chemoprevention of squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1920. doi:10.1158/1538-7445.AM2015-1920

Abstract 815: Integrated genomic analysis of cutaneous squamous cell carcinoma progression.

Skin cancer is the most common class of malignancy in humans. In the United States, there are over 3 million cases of skin cancer a year with significant costs not only in morbidity and mortality, but an estimated

203 A proteome-transcriptome-miRnome integratead analysis identifies similarity between UV-exposed skin and wounding skin

500 million in treatment-related costs and

098 A cohort of miRNAs can be used as an early predictive biomarker of UV-driven cutaneous squamous cell carcinoma

2 billion in overall economic impact including lost productivity. The vast majority of these skin cancers arise in keratinocytes and within this group, cutaneous squamous cell carcinoma (cSCC) comprises 15-20% of cases. cSCC has the best-defined progression from a distinct precancerous lesion, the actinic keratosis (AK) to invasive cSCC which has appreciable metastatic potential particularly in high-risk scenarios. Beyond serial, disfiguring surgeries, there is no effective therapy. AKs are the most common precancerous lesion in humans, affecting upwards of 5.5% of women and 13.9% of men in U.S. Our understanding of the molecular events that lead from normal skin to AK to cSCC is very poor and represents a fundamental gap in our understanding of this progression sequence. Such an understanding would be of enormous importance. While destructive modalities are the mainstay of treatment of AK, they must be repetitively used and there is no basis for identifying those most likely to progress. Therefore, there is a tremendous need for rationally designed targeted diagnostics and therapy for AKs, which presents an ideal opportunity for molecularly-informed skin cancer screening and secondary prevention. We have performed next generation sequencing (Illumina HiSeq) for microRNA and mRNA on matched samples from 10 patients. Each matched set is composed of clinically normal skin, AK, and cSCC from the same patient, minimizing background genetic heterogeneity. For miRNA-seq, 6.1 million reads with 720,000 mapped reads were obtained on average. RNA-seq analysis yielded over 64 million reads per sample with 92% mapped. Following unsupervised clustering, we found cSCCs clustered together, and AKs clustered with both normal skin and with cSCC confirming the clinico-pathologic characterization of these lesions as precursors to cSCC. Changes in expression of only two miRNA and 367 mRNA emerged as the most significant predictors of overall progression from normal skin to AK to cSCC. In pairwise comparisons between normal skin and cSCC, we identified 628 functional miRNA-mRNA pairs comprising a total of 59 miRNA and 179 predicted mRNA targets. Surprisingly, the number of miRNA and mRNA expression changes between AK and cSCC were very limited ( Citation Format: Vida Chitsazzadeh, Weimin Xiao, Preethi Gunaratne, Cristian Coarfa, Xiaoping Su, Tri H. Nguyen, Valencia D. Thomas, Aaron K. Joseph, Elsa R. Flores, Kenneth Y. Tsai. Integrated genomic analysis of cutaneous squamous cell carcinoma progression. [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 815. doi:10.1158/1538-7445.AM2013-815