Hepatocellular carcinoma (HCC) remains a global health challenge with high mortality rates, largely due to late diagnosis and suboptimal efficacy of current therapies. With the imperative need for more reliable, non-invasive diagnostic tools and novel therapeutic strategies, this study focuses on the discovery and application of novel genetic biomarkers for HCC using explainable artificial intelligence (XAI). Despite advances in HCC research, current biomarkers like Alpha-fetoprotein (AFP) exhibit limitations in sensitivity and specificity, necessitating a shift towards more precise and reliable markers. This paper presents an innovative multi-model XAI and a probabilistic causal inference framework to identify and validate key genetic biomarkers for HCC prognosis. Our methodology involved analyzing clinical and gene expression data to identify potential biomarkers with prognostic significance. The study utilized robust AI models validated against extensive gene expression datasets, demonstrating not only the predictive accuracy but also the clinical relevance of the identified biomarkers through explainable metrics. The findings highlight the importance of biomarkers such as TOP3B, SSBP3, and COX7A2L, which were consistently influential across multiple models, suggesting their role in improving the predictive accuracy for HCC prognosis beyond AFP. Notably, the study also emphasizes the relevance of these biomarkers to the Hispanic population, aligning with the larger goal of demographic-specific research. The application of XAI in biomarker discovery represents a significant advancement in HCC research, offering a more nuanced understanding of the disease and laying the groundwork for improved diagnostic and therapeutic strategies.

Background:The incidence and mortality rates of hepatocellular carcinoma (HCC) among Hispanics in the United States are much higher than those of non-Hispanic whites. We conducted comprehensive multi-omics analyses to understand molecular alterations in HCC among Hispanic patients. Methods:Paired tumor and adjacent non-tumor samples were collected from 31 Hispanic HCC in South Texas (STX-Hispanic) for genomic, transcriptomic, proteomic, and metabolomic profiling. Additionally, serum lipids were profiled in 40 Hispanic and non-Hispanic patients with or without clinically diagnosed HCC. Results:Exome sequencing revealed high mutation frequencies of AXIN2 and CTNNB1 in STX Hispanic HCCs, suggesting a predominant activation of the Wnt/β-catenin pathway. The TERT promoter mutation frequency was also remarkably high in the Hispanic cohort. Cell cycles and liver functions were identified as positively- and negatively-enriched, respectively, with gene set enrichment analysis. Gene sets representing specific liver metabolic pathways were associated with dysregulation of corresponding metabolites. Negative enrichment of liver adipogenesis and lipid metabolism corroborated with a significant reduction in most lipids in the serum samples of HCC patients. Two HCC subtypes from our Hispanic cohort were identified and validated with the TCGA liver cancer cohort. The subtype with better overall survival showed higher activity of immune and angiogenesis signatures, and lower activity of liver function-related gene signatures. It also had higher levels of immune checkpoint and immune exhaustion markers. Conclusions:Our study revealed some specific molecular features of Hispanic HCC and potential biomarkers for therapeutic management of HCC and provides a unique resource for studying Hispanic HCC.

Hispanics in the US experience a disproportionate burden of hepatocellular carcinoma (HCC) with an overall incidence and cancer-related mortality nearly two times higher than non-Hispanic whites. The genomic characteristics and underlying molecular mechanisms driving tumorigenesis associated with HCC in Hispanic patients remain unknown. Here, we used 31 paired tumor and adjacent non-tumor liver samples from Hispanic patients in South Texas to perform integrated genomic, transcriptomic, proteomic, and metabolomic analyses. Similar to HCC in other ethnic groups, CTNNB1 and TP53 were found to have the most frequent somatic mutations in the Hispanic HCC. On the other hand, AXIN2 and to a lesser degree MTOR appear more frequently mutated in the Hispanics compared to the various ethnic groups included in The Cancer Genome Atlas (TCGA) study. Somatic mutations in the Hispanic HCC showed signatures of tobacco chewing and aflatoxin exposure, which are different from the signatures found frequently in the liver cancer patients in the TCGA study. The WNT, TP53, & cell cycle were the most frequently altered oncogenic pathways in our cohort. Unsupervised clustering of patients, based on the enrichment scores from single sample Gene Set Enrichment Analysis (ssGSEA) using our tumor and nontumor paired RNA-Seq and proteomic data, revealed two clusters with striking differences in various signaling pathways and cellular functions. A similar clustering pattern with a significant difference in overall survival was seen in the patients from the TCGA study. Irrespective of ethnicity, immune infiltration in the tumor microenvironment and liver function-related pathways were significantly different between patients in these clusters. Integrated transcriptomic, proteomic, and metabolomic analyses identified significant negative enrichments in gluconeogenesis, TCA cycle, and glutamate metabolism in Hispanic HCC. These results suggest the existence of molecular mechanisms either unique in Hispanics or similar to non-Hispanic HCC, especially from the TCGA study. Our findings might provide insights to developing predictive biomarkers for the early diagnosis and novel therapeutics for the management of HCC. Citation Format: Debodipta Das, Xiaojing Wang, Yu-Chiao Chiu, Hakim Bouamar, Yidong Chen, Siyuan Zheng, Francisco G. Cigarroa, Lu-Zhe Sun. Comprehensive multi-omic characterization of hepatocellular carcinoma in Hispanic patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3175.

Introduction: The cell cycle inhibitor palbociclib has been shown to have a cytostatic effect on hepatocellular carcinoma (HCC) cells via the induction of quiescence and senescence, though it has limited cytocidal effect. We hypothesized that arrested HCC cells upregulate mTOR signaling to stay metabolically active and investigated the effect of combining palbociclib with the mTOR inhibitor rapamycin on preclinical models of HCC. Methods: We identified commonly altered molecular markers in HCC, including cell cycle related genes, using single sample gene set enrichment analysis (ssGSEA) of 13 paired (tumor and adjacent non-tumor) RNA-seq and 15 paired proteomic data sets as well as 50 paired RNA-seq data sets from TCGA. Subsequently, we studied the in-vitro effects of palbociclib and rapamycin by evaluating induction of senescence, inhibition of cell growth with fixed ratio combination treatment as well as Western Blotting to establish mechanistic effects. We tested the in-vivo anti-tumor efficacy of palbociclib plus rapamycin in athymic nude mice with growing xenograft tumors. We then generated immunocompetent murine HCC models using hydrodynamic transfection of oncogenes ΔN90-β-catenin and c-Met, and measured plasma AFP levels as a marker of HCC to assess the efficacy of treatment with palbociclib and/or rapamycin. Results: ssGSEA revealed significant positive enrichment of several cell cycle related gene sets in all our HCC cases. Palbociclib treatment in vitro showed an increase in senescence associated β-galactosidase staining as well as increased levels of intrinsic CDK inhibitors p16, p15, p21 and p27, indicating senescence. Fixed ratio treatment with palbociclib and rapamycin established a synergistic interaction between the drugs, as indicated by a combination index of less than 1. We also observed an increase in levels of proteins of the mTOR pathway (p-mTOR, p-4eBP1, p-p70 and p-s6) after palbociclib treatment, followed by suppression of the levels on addition of rapamycin. In the xenograft HCC model, combination treatment with palbociclib and rapamycin resulted in slower tumor growth and significantly increased time to critical tumor volume (2000 mm3) (log rank, p <0.001). In the hydrodynamic transfection HCC model, we observed a significantly lower mean AFP level in the combination treatment group, along with lower liver weights which were closer to the liver weights of normal mice. Conclusion: HCC cells overexpress genes and proteins that drive cell cycle progression. Palbociclib induces senescence in HCC cells along with an associated upregulation in mTOR signaling, and combination with rapamycin has a synergistic effect on inhibition of tumor growth in vitro as well as in vivo, possibly by abrogating both actively growing and quiescent/senescent HCC cells. The combination of the two drugs may have potential as a novel therapy for HCC. Citation Format: Ankur Tiwari, Araceli Huerta, Hakim Bouamar, Debodipta Das, Francisco Cigarroa, LuZhe Sun. Combination of CDK 4/6 inhibitor palbociclib with rapamycin synergistically inhibits growth of hepatocellular carcinoma in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2679.

Hepatocellular carcinoma (HCC) is the major type of liver cancer and one of the few cancers with a steady increase in incidence and mortality rate over the past several decades in the USA. It disproportionately affects Hispanics in the USA with an incidence and mortality rate about two to three times higher in Hispanics than in non-Hispanic whites. To identify molecular features associated with the Hispanic HCC we have generated genome-wide sequencing (i.e., exome-seq, RNA-seq) data and mass spectrometry-based proteomics data from HCC tumor and matched blood and/or adjacent normal tissues of Hispanic patients based in South Texas. The landscape of somatic mutation in HCC tumors revealed the aflatoxin-induced mutational signature (COSMIC) in some patients indicating an association of aflatoxin mediated toxicity in them. Similar to The Cancer Genome Atlas (TCGA) study, with predominant non-Hispanic HCC tumors, we could identify the most frequent somatic mutations in CTNNB1 and TP53. Other frequently (>10%) mutated genes in our patients appear to be mutated at relatively lower rate in the TCGA patients. Mutation(s) in some of the frequently mutated genes might contribute to the significant enrichment of cellular signaling pathways observed from the Gene Set Enrichment Analysis (GSEA) using paired RNA-seq data. Single sample GSEA (ssGSEA) of our RNA-seq and proteomic data sets revealed two clusters of HCC tumors, denoted as H1 and H2 clusters, with striking differences in various signaling pathways and cellular functions. Patients from the H1 cluster and two sub-clusters within the H2 cluster showed a significant difference in their survival rate based on overall survival information from the TCGA study. The transcriptomic data from Hispanic & non-Hispanic HCC identified activated immune evasion mechanisms in H1 patients. Sorafenib treatment was found to be associated with a lower hazard ratio for overall survival in patients from the H1 cluster compared to the H2 cluster. In sum, we have identified genes more frequently mutated in Hispanic HCC than in non-Hispanic HCC, and cellular and tissue functions significantly enriched only in the Hispanic HCC. We also report a novel classification of HCC tumors with significant differences in various cellular signaling pathways and overall survival. Our findings might be used as biomarkers for the early prediction and better therapeutic management of HCC. Citation Format: Debodipta Das, Xiaojing Wang, Yu-Chiao Chiu, Hakim Bouamar, Yidong Chen, Siyuan Zheng, Francisco G. Cigarroa, Lu-Zhe Sun. Integration of multi-omics data reveals molecular features of Hispanic hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2272.

Oral squamous cell carcinoma of the gingivo-buccal region (OSCC-GB) accounts for the highest cancer morbidity and mortality among men in India. It has been observed that about one-third of individuals with oral leukoplakia, a dysplastic precancerous lesion in the oral cavity, progress to oral cancer. We aimed to identify systematic transcriptomic changes as a normal tissue in the oral cavity progresses to frank OSCC-GB. Seventy-two OSCC-GB patients, from multiple hospitals, were recruited, and transcriptome analysis of tumor and adjacent normal tissue (of all patients) and adjacent leukoplakia tissue (of a subset of 25 unselected patients with concomitant leukoplakia) was performed. We have identified many differences in the transcriptomic profiles between OSCC-GB and squamous cell carcinoma of the head and neck regions. Compared to the normal/precancerous tissue, significant enrichment of ECM−receptor interaction, PI3K-Akt signaling, cytokine−cytokine receptor interaction, focal adhesion, and cell cycle pathways were observed in OSCC-GB. Using gene set enrichment analysis, we identified a profound role of interferon receptor signaling in tumor growth by activating immune evasion mechanisms. The role of tumor-infiltrating immune cells further supported the growth and immunosuppressive mechanism of tumor tissues. Some immune evasion genes— CD274, CD80 , and IDO1 —were found to be activated even in the precancerous tissue. Taken together, our findings provide a clear insight into the sequential genetic dysregulation associated with progression to oral cancer. This insight provides a window to the development of predictive biomarkers and therapeutic targets for gingivo-buccal oral cancer.

Although the Sonic hedgehog (SHH) signaling pathway has been implicated in promoting malignant phenotypes of prostate cancer, details on how it is activated and exerts its oncogenic role during prostate cancer development and progression is less clear. Here, we show that GLI3, a key SHH pathway effector, is transcriptionally upregulated during androgen deprivation and posttranslationally stabilized in prostate cancer cells by mutation of speckle-type POZ protein (SPOP). GLI3 is a substrate of SPOP-mediated proteasomal degradation in prostate cancer cells and prostate cancer driver mutations in SPOP abrogate GLI3 degradation. Functionally, GLI3 is necessary and sufficient for the growth and migration of androgen receptor (AR)–positive prostate cancer cells, particularly under androgen-depleted conditions. Importantly, we demonstrate that GLI3 physically interacts and functionally cooperates with AR to enrich an AR-dependent gene expression program leading to castration-resistant growth of xenografted prostate tumors. Finally, we identify an AR/GLI3 coregulated gene signature that is highly correlated with castration-resistant metastatic prostate cancer and predictive of disease recurrence. Together, these findings reveal that hyperactivated GLI3 promotes castration-resistant growth of prostate cancer and provide a rationale for therapeutic targeting of GLI3 in patients with castration-resistant prostate cancer (CRPC). Implications: We describe two clinically relevant mechanisms leading to hyperactivated GLI3 signaling and enhanced AR/GLI3 cross-talk, suggesting that GLI3-specific inhibitors might prove effective to block prostate cancer development or delay CRPC.

Background: Studies of epigenomic alterations associated with diseases primarily focus on methylation profiles of promoter regions of genes, but not of other genomic regions. In our past work (Das et al. 2019) on patients suffering from gingivo-buccal oral cancer – the most prevalent form of cancer among males in India – we have also focused on promoter methylation changes and resultant impact on transcription profiles. Here, we have investigated alterations in non-promoter (gene-body) methylation profiles and have carried out an integrative analysis of gene-body methylation and transcriptomic data of oral cancer patients. Methods: Tumor and adjacent normal tissue samples were collected from 40 patients. Data on methylation in the non-promoter (gene-body) regions of genes and transcriptome profiles were generated and analyzed. Because of high dimensionality and highly correlated nature of these data, we have used Random Forest (RF) and other data-analytical methods. Results: Integrative analysis of non-promoter methylation and transcriptome data revealed significant methylation-driven alterations in some genes that also significantly impact on their transcription levels. These changes result in enrichment of the Central Carbon Metabolism (CCM) pathway, primarily by dysregulation (overexpression) of (a) NTRK3 , which plays a dual role as an oncogene and a tumor suppressor; (b) SLC7A5 ( LAT1 ) which is a transporter dedicated to essential amino acids, and is overexpressed in cancer cells to meet the increased demand for nutrients that include glucose and essential amino acids; and, (c) EGFR which has been earlier implicated in progression, recurrence, and stemness of oral cancer, but we provide evidence of epigenetic impact on overexpression of this gene for the first time. Conclusions: In rapidly dividing cancer cells, metabolic reprogramming from normal cells takes place to enable enhanced proliferation. Here, we have identified that among oral cancer patients, genes in the CCM pathway – that plays a fundamental role in metabolic reprogramming – are significantly dysregulated because of perturbation of methylation in non-promoter regions of the genome. This result compliments our previous result that perturbation of promoter methylation results in significant changes in key genes that regulate the feedback process of DNA methylation for the maintenance of normal cell division.

Background: Studies of epigenomic alterations associated with diseases primarily focus on methylation profiles of promoter regions of genes, but not of other genomic regions. In our past work (Das et al. 2019) on patients suffering from gingivo-buccal oral cancer – the most prevalent form of cancer among males in India – we have also focused on promoter methylation changes and resultant impact on transcription profiles. Here, we have investigated alterations in non-promoter (gene-body) methylation profiles and have carried out an integrative analysis of gene-body methylation and transcriptomic data of oral cancer patients. Methods: Tumor and adjacent normal tissue samples were collected from 40 patients. Data on methylation in the non-promoter (gene-body) regions of genes and transcriptome profiles were generated and analyzed. Because of high dimensionality and highly correlated nature of these data, we have used Random Forest (RF) and other data-analytical methods.Results: Integrative analysis of non-promoter methylation and transcriptome data revealed significant methylation-driven alterations in some genes that also significantly impact on their transcription levels. These changes result in enrichment of the Central Carbon Metabolism (CCM) pathway, primarily by dysregulation (overexpression) of (a) NTRK3, which plays a dual role as an oncogene and a tumor suppressor; (b) SLC7A5 (LAT1) which is a transporter dedicated to essential amino acids, and is overexpressed in cancer cells to meet the increased demand for nutrients that include glucose and essential amino acids; and, (c) EGFR which has been earlier implicated in progression, recurrence, and stemness of oral cancer, but we provide evidence of epigenetic impact on overexpression of this gene for the first time.Conclusions: In rapidly dividing cancer cells, metabolic reprogramming from normal cells takes place to enable enhanced proliferation. Here, we have identified that among oral cancer patients, genes in the CCM pathway – that plays a fundamental role in metabolic reprogramming – are significantly dysregulated because of perturbation of methylation in non-promoter regions of the genome. This result compliments our previous result that perturbation of promoter methylation results in significant changes in key genes that regulate the feedback process of DNA methylation for the maintenance of normal cell division.

BACKGROUND:Gingivo-buccal oral squamous cell carcinoma (OSCC-GB) is the most common cancer among men in India and is associated with high mortality. Although OSCC-GB is known to be quite different from tongue cancer in its genomic presentation and its clinical behavior, it is treated identically as tongue cancer. Predictive markers of prognosis and therapy that are specific to OSCC-GB are, therefore, required. Although genomic drivers of OSCC-GB have been identified by whole exome and whole genome sequencing, no epigenome-wide study has been conducted in OSCC-GB; our study has filled this gap, and has discovered and validated epigenomic hallmarks of gingivobuccal oral cancer.METHODS:We have carried out integrative analysis of epigenomic (n = 87) and transcriptomic (n = 72) profiles of paired tumor-normal tissues collected from OSCC-GB patients from India. Genome-wide DNA methylation assays and RNA-sequencing were performed on high-throughput platforms (Illumina) using a half-sample of randomly selected patients to discover significantly differentially methylated probes (DMPs), which were validated on the remaining half-sample of patients.RESULTS:About 200 genes showed significant inverse correlation between promoter methylation and expression, of which the most significant genes included genes that act as transcription factors and genes associated with other cancer types. Novel findings of this study include identification of (a) potential immunosuppressive effect in OSCC-GB due to significant promoter hypomethylation driven upregulation of CD274 and CD80, (b) significant dysregulation by epigenetic modification of DNMT3B (upregulation) and TET1 (downregulation); and (c) known drugs that can reverse the direction of dysregulation of gene expression caused by promoter methylation.CONCLUSIONS:In OSCC-GB patients, there are significant alterations in expression of key genes that (a) regulate normal cell division by maintenance of balanced DNA methylation and transcription process, (b) maintain normal physiological signaling (PPAR, B cell receptor) and metabolism (arachidonic acid) pathways, and (c) provide immune protection against antigens, including tumor cells. These findings indicate novel therapeutic targets, including immunotherapeutic, for treatment of OSCC-GB.

Purpose. To identify the spectrum of somatic mutations in an Asian Indian patient with uveal melanoma (UM) without metastasis using exome sequencing.Case Report. A 49-year-old man from India was diagnosed as having cilio-choroidal (uveal) melanoma (UM), without metastasis, in his right eye with the help of magnetic resonance imaging. This was later confirmed by histopathological evaluation. Two individuals from India with non-neoplastic blind eyes were recruited as controls. The affected eyes from the UM patient and the two control individuals were enucleated, and uveal tissues were collected. DNA was extracted from uveal tissue, and the matched blood sample from each of the three individuals was followed by exome sequencing. Statistical and bioinformatic analyses were done to identify somatic mutations and their putative associations with UM. Thirty-one somatic mutations (25 amino acid altering) in protein-coding (exonic) regions were detected in the UM patient. Of the amino acid-altering somatic mutations, 16 mutations were predicted to be candidate mutations relevant to UM. Somatic mutations, putatively causal for UM, were identified in GNAQ, SF3B1, and SOX10.Conclusions. Somatic mutations in GNAQ and SF3B1 genes were probable drivers of UM in the Indian patient; these were also reported earlier in some White patients. In addition, a frameshift deletion of 20 base pairs has been identified in SOX10 in the UM patient. Somatic mutations in SOX10, a transcription factor, which acts upstream of microphthalmia-associated transcription factor and synergizes with microphthalmia-associated transcription factor, was identified in some melanoma cell lines. The transcription factor SOX10 was found to have an essential role in melanocyte development and pigmentation. Our finding of the frameshift deletion (p. H387fs) in exon 4 of SOX10 in UM provides an important insight and complements earlier findings of mutations in GNAQ and SF3B1 on the genomic basis of UM.