Tran N. Nguyen

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.

REST represses miR-124 and miR-203 to regulate distinct oncogenic properties of glioblastoma stem cells

Background Glioblastoma (GBM) is one of the most common, aggressive, and invasive human brain tumors. There are few reliable mechanism-based therapeutic approaches for GBM patients. The transcriptional repressor RE1 silencing transcriptional factor (REST) regulates the oncogenic properties of a class of GBM stem-like cells (high-REST [HR]-GSCs) in humans. However, it has been unclear whether REST represses specific targets to regulate specific oncogenic functions or represses all targets with overlapping functions in GSCs. Methods We used genome-wide, biochemical, and mouse intracranial tumorigenic assays to identify and determine functions of microRNA (miR) targets of REST in 2 independent HR-GSC lines. Results Here we show that REST represses 2 major miR gene targets in HR-GSCs: miR-203, a new target, and miR-124, a known target. Gain of function of miR-124 or miR-203 in HR-GSCs increased survival in tumor-bearing mice. Importantly, the increased survival of tumor-bearing mice caused by knockdown of REST in HR-GSCs was reversed by double knockdown of REST and either miR-203 or miR-124, indicating that these 2 miRs are critical tumor suppressors that are repressed in REST-mediated tumorigenesis. We further show that while miR-124 and the REST-miR-124 pathways regulate self-renewal, apoptosis and invasion, miR-203 and the REST-miR-203 pathways regulate only invasion. We further identify and validate potential mRNA targets of miR-203 and miR-124 in REST-mediated HR-GSC tumor invasion. Conclusions These findings indicate that REST regulates its miR gene targets with overlapping functions and suggest how REST maintains oncogenic competence in GSCs. These mechanisms could potentially be utilized to block REST-mediated GBM tumorigenesis.

APOBEC mutation drives early-onset squamous cell carcinomas in recessive dystrophic epidermolysis bullosa

Early-onset squamous cell carcinoma in recessive dystrophic epidermolysis bullosa patients is characterized by APOBEC mutagenesis. Mutational signature sleuthing Individuals with the inherited skin disease recessive dystrophic epidermolysis bullosa (RDEB) are predisposed to developing aggressive squamous cell carcinomas (SCCs), although why this patient group is prone to these cancers at such early ages is unknown. Cho et al. sequenced multiple RDEB SCC tumors and found that the mutation profile in these carcinomas was most consistent with APOBEC-associated mutagenesis, unlike other types of SCC that may be driven by ultraviolet light or tobacco smoke exposure. This finding could open up new lines of thinking on how to successfully prevent or target SCCs in RDEB patients. Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin and mucous membrane fragility disorder complicated by early-onset, highly malignant cutaneous squamous cell carcinomas (SCCs). The molecular etiology of RDEB SCC, which arises at sites of sustained tissue damage, is unknown. We performed detailed molecular analysis using whole-exome, whole-genome, and RNA sequencing of 27 RDEB SCC tumors, including multiple tumors from the same patient and multiple regions from five individual tumors. We report that driver mutations were shared with spontaneous, ultraviolet (UV) light–induced cutaneous SCC (UV SCC) and head and neck SCC (HNSCC) and did not explain the early presentation or aggressive nature of RDEB SCC. Instead, endogenous mutation processes associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide–like (APOBEC) deaminases dominated RDEB SCC. APOBEC mutation signatures were enhanced throughout RDEB SCC tumor evolution, relative to spontaneous UV SCC and HNSCC mutation profiles. Sixty-seven percent of RDEB SCC driver mutations was found to emerge as a result of APOBEC and other endogenous mutational processes previously associated with age, potentially explaining a >1000-fold increased incidence and the early onset of these SCCs. Human papillomavirus–negative basal and mesenchymal subtypes of HNSCC harbored enhanced APOBEC mutational signatures and transcriptomes similar to those of RDEB SCC, suggesting that APOBEC deaminases drive other subtypes of SCC. Collectively, these data establish specific mutagenic mechanisms associated with chronic tissue damage. Our findings reveal a cause for cancers arising at sites of persistent inflammation and identify potential therapeutic avenues to treat RDEB SCC.

Abstract 406: A proteome-transcriptome-miRnome integrated analysis identifies similarity between UV-exposed skin and wounding skin

Cutaneous squamous cell carcinoma (cuSCC) is the second most common skin cancer, for which long term UV exposure and chronic wounding are the dominant risk factors. Despite these clinically established connections, little is understood about the early molecular response of human skin to UV exposure and its connection to acute wounding and cuSCC. Thus, our goal is to find common and specific signatures driven by UV-exposure and wounding as a means of developing new approaches for treating and preventing cuSCC. Here, we perform integrated analyses of proteomic, RNA-seq and miR-seq on 3 datasets: (1) UV-unexposed and acute UV-exposed human skin, (2) public dataset on acute wound healing and (3) our previously published dataset on normal skin and cuSCC from humans. We find that biological signatures and processes regulated by acute UV exposure and wounding has profound similarity. miR-seq data shows that miR-223, miR-132 and miR-142 are significantly upregulated in both acute events. Combined gene set enrichment analysis shows that G-protein-coupled-receptors (GPCRs) pathways are upregulated, possibly through Gαi activation. While ECM remodeling is significantly enriched in all three datasets, gene expression regulated by PPARα is suppressed. Interestingly, upregulation of matrisome components is observed among all three datasets. This suggests that these changes are important early events that regulated by both UV-exposure and wounding which eventually can promote cuSCC initiation. Thus, our findings suggest that these common signatures can be potentially validated as chemopreventive targets for cuSCC. Citation Format: Tran N. Nguyen, Kimal Rajapakshe, Stanislav Avdieiev, Courtney Nicholas, Vida Chitsazzadeh, Eric Welsh, Bin Fang, John Koomen, Cristian Coarfa, Janine Einspahr, Kenneth Y. Tsai. A proteome-transcriptome-miRnome integrated analysis identifies similarity between UV-exposed skin and wounding skin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 406.

Abstract 5599: Soluble PD-L1 as a surrogate biomarker of metastatic progression and resistance to antiangiogenic therapy

Immune-checkpoint inhibitors are now approved for the treatment of early- and late-stage cancers. These include agents that block the T-cell regulatory protein programmed cell death 1 (PD-1) from being activated by the PD-1 ligand 1 (PD-L1) expressed on cancer cells. There is an urgent need to identify biomarkers of PD-1 pathway inhibition that would predict patient populations responsive to treatment and/or serve as surrogates for drug activity and resistance. PD-L1 expression on tumors is currently a biomarker candidate, but reliable detection and quantification methodologies have proven challenging to standardize. Recently, a soluble PD-L1 (sPD-L1) fragment was identified that can derive from cell-bound PD-L1. Retrospective clinical examinations of sPD-L1 levels in cancer patients suggest a potential use as a surrogate for disease progression and response to treatment; but few preclinical studies have been performed to test this predictive value. We undertook experiments to evaluate plasma sPD-L1 in mouse tumor models during localized primary tumor growth (after orthotopic cell implantation) and spontaneous metastatic disease progression (after surgical removal of the primary). Mouse syngeneic and human xenograft implantation models included breast, kidney, colon, and melanoma cell systems. Our results show that circulating plasma sPD-L1 can correlate with primary and metastatic progression in a stage and model specific manner. Next, we evaluated sPD-L1 following treatment with neutralizing antibodies to PD-1 and PD-L1 in tumor-free mice and found significant dose-dependent sPD-L1 increases, suggesting systemic changes may have utility as a measurement of target saturation and dosing independent of tumor growth. Finally, with current approvals of PD-1 inhibitors in renal cell carcinoma (RCC) patients previously treated with antiangiogenic agents that block vascular endothelial growth factor (VEGF), we evaluated plasma in mouse models of sunitinib resistance - a VEGF receptor tyrosine kinase inhibitor (RTKI). Our results demonstrate that VEGF pathway resistance yields changes in sPD-L1 and may be useful in predicting response to PD-1 pathway inhibition in the refractory setting. Together, these investigations suggest that circulating sPD-L1 changes during disease progression (both local and disseminated) may serve as a potential predictive biomarker for immune-checkpoint and antiangiogenic therapy. Citation Format: Michalis Mastri, Amanda Tracz, Yuhao Shi, Georg Bjarnason, Tran Nguyen, Brian Rini, John M.L. Ebos. Soluble PD-L1 as a surrogate biomarker of metastatic progression and resistance to antiangiogenic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5599. doi:10.1158/1538-7445.AM2017-5599

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 864: ANG4043: A new brain-penetrant peptide-antibody conjugate that reduces tumor growth in a HER2-positive orthotopic tumor model.

While monoclonal antibodies to receptor tyrosine kinases such as HER2 have been demonstrated to reduce tumor size and increase survival, these agents achieve little to no brain penetration, making them ineffective against metastatic brain tumors. The blood-brain barrier (BBB), which restricts entry of proteins such as mAbs into the brain, is comprised of capillary endothelial cells with tight junctions and efflux pumps. Brain entry of nutrients, hormones, and other required molecules is accomplished by processes such as receptor-mediated transcytosis. As low-density lipoprotein receptor-related protein 1 (LRP1) is known to perform this function in BBB endothelial cells, we have created a family of peptides (Angiopeps) designed for LRP1 recognition. These proprietary Angiopeps can be used to create novel Peptide-Drug Conjugates that successfully cross the BBB by LRP1-mediated transcytosis. Here we describe chemical conjugation of Angiopep-2 (An2) to a mAb against HER2. This Peptide-Antibody Conjugate, ANG 4043, displays HER2 binding affinity and in vitro cytotoxic potency similar to that of native anti-HER2. ANG4043 demonstrates a high rate of entry into the brain, consistent with achieving therapeutic concentrations. The plasma half-life of ANG4043 is similar to that of Anti-HER2 in mouse. Mice with intracranially implanted BT-474 cells showed reduced brain tumor size when dosed with ANG4043 compared to controls. Overall, these data demonstrate that a brain-penetrant Peptide-Antibody Conjugate is efficacious in a mouse HER2-positive tumor model. These results extend the validation of An2 conjugation beyond small molecules and peptides to include larger molecules such as therapeutic mAbs for development of new brain-penetrant antitumor therapeutics. Citation Format: Jean E. Lachowicz, Michel Demeule, Christian Che, Sasmita Tripathy, Jean-Christophe Currie, Anthony Regina, Tran Nguyen, Simon Lord-Dufour, Jean-Paul Castaigne. ANG4043: A new brain-penetrant peptide-antibody conjugate that reduces tumor growth in a HER2-positive orthotopic tumor model. [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 864. doi:10.1158/1538-7445.AM2013-864