Loss of TIP30 Accelerates Pancreatic Cancer Progression and Metastasis

Indiana University-Purdue University Indianapolis (IUPUI)Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth leading cause of cancer-related death in the United States, and is characterized by key driver mutations (e.g. KRAS, TP53, CDKN2A, and SMAD4), elevated expression of growth factors such as TGF-βs and the EGF receptor (EGFR), a markedly desmoplastic stroma, and a propensity to develop multi-organ metastases and chemoresistance. Consistent with its aggressive nature, the 5-year survival rate for PDAC is 8-9%, which demonstrates an urgent need to develop novel therapies. High expression levels of microRNA-10b (miR-10b) in PDAC tissues are associated with decreased patient survival and earlier appearance of metastatic disease following neoadjuvant chemoradiotherapy. miR-10b downregulates the expression of transcription coactivator Tat-Interacting Protein 30 (TIP30) by targeting its 3’UTR. TIP30 has multiple reported functions. TIP30 suppresses tumor formation and metastasis, forms a complex that regulates EGFR trafficking and degradation, and transcriptionally upregulates pro-apoptotic genes. Alterations in TIP30 have been reported in multiple human cancers, including pancreatic cancer. We hypothesized that Tip30-deficiency accelerates PDAC progression and metastasis in a murine model of PDAC. To test this hypothesis, we crossed mice with oncogenic Kras (KC) localized to the pancreas epithelium, with Tip30-deficient mice (K30C). We compared PDAC histopathology between Tip30-heterozygous (K30+/-C) and Tip30-null (K30-/-C) mice. Tip30-heterozygosity accelerated PDAC-lesion-associated pancreatic cancer cell (PCC) pulmonary seeding. By contrast, total loss of Tip30 enhanced PCC micrometastatic seeding to the liver and hepatic metastasis. K30+/-C mice also presented with an early, increased penetrance of lung lesions and lung adenocarcinoma; and PCCs isolated from K30+/-C pancreata exhibited increased EGFR protein levels. These findings suggest that TIP30 deficiency can have a dose-dependent effect on organotropic metastasis and EGFR levels in PCCs. Future studies will delineate the molecular consequences of TIP30 loss in PDAC and contribute to a broader understanding of pancreatic cancer metastasis.2020-08-0

Metastatic Progression and Tumour Heterogeneity

Improved understanding of the cellular and molecular makeup of tumors in the last 30 years has unraveled a previously unexpected level of heterogeneity among tumor cells as well as within the tumor microenvironment. The concept of tumor heterogeneity underlines the realization that different tumors can display significant differences in their genomic content as well as in their overall behavior. Our capacity to better understand the heterogeneous make up of tumors has very important consequences on our ability to design efficient therapeutic strategies to improve patient survival. This book highlights several aspects of tumor heterogeneity in the context of metastatic development and summarize some of the challenges posed by heterogeneity for tumor diagnostics and therapeutic management of tumors

Investigating the Importance of Physiological Cell Culture Conditions in Modeling Cancer Metabolism and Metabolic Reprogramming

Standard cell culture conditions do not mimic the physiological environment of cancer cells. Traditional culture media contain metabolites at concentrations that far exceed conditions measured in vivo, and oxygen is often unregulated, exposing cells to atmospheric oxygen concentrations (~18%), rather than the 0-3% O2 measured in solid tumours in vivo. Recently, plasma-like media have been developed to address these limitations, aiming to improve culture conditions and maintain biologically relevant cancer phenotypes in vitro. However, these conditions remain unrepresentative of interstitial fluid in solid tumours. The goal of this thesis was to investigate how physiological culture conditions affect cancer cell behaviours, specifically cell metabolism and adaptive metabolic responses. In the first data chapter of this thesis, I explored nutrient exhaustion in physiological plasma-like medium (Plasmax) at 18% and 5% O2, and the adaptive mechanisms by which cancer cells can maintain survival under metabolic stress conditions. Here, I found that glucose and amino acid depletion from Plasmax over 48 hours is associated with several adaptive mechanisms consistent with metabolic reprogramming in vivo. Given these responses, I hypothesized that a media formulation designed using metabolite concentrations from tumour interstitial fluid may modulate metabolic phenotypes in a similar manner, providing a more physiologically relevant culture model for cancerous cells. Data chapter 2 addresses this hypothesis, whereby I formulated a novel cell culture medium using quantitative metabolite data from murine pancreatic ductal adenocarcinoma (PDAC) tumour interstitial fluid, named Tumour Microenvironment Medium (TMEM), and investigated the effects of TMEM and 1.5% O2 on an adapted murine PDAC cell line (KPCY). Importantly, I found that tumour-like conditions elicited a substantial transcriptional and functional response in cultured cells, modulating cell proliferation, migration, glucose utilization, and mitochondrial bioenergetics in ways relevant to in vivo cancer biology. Overall, the results of this thesis highlight the responsiveness of cultured cells to their environment, and the importance of representative culture conditions in the acquisition of biologically accurate experimental data

Molecular characterization and validation of new biomarkers for early detection of familial pancreatic cancer

Está previsto que el adenocarcinoma ductal pancreático (PDAC) se convierta en la segunda causa principal de muerte relacionada con el cáncer para 2030. Las bajas tasas de supervivencia se deben principalmente a la ausencia de síntomas específicos y al diagnóstico tardío, con solo el 20% de los tumores siendo candidatos a una resección potencialmente curativa, aunque con una alta tasa de recurrencia. Los antecedentes familiares y la predisposición genética son los factores de riesgo no modificables más importantes, con un riesgo de PDAC mayor según aumenta el número de familiares afectados. Este subgrupo representa hasta el 15% del total de casos diagnosticados de PDAC. Algunas familias presentan mutaciones germinales en genes de susceptibilidad al cáncer bien conocidos, como BRCA2, CDKN2A, STK11 o MLH1, y se clasifican como cáncer de páncreas hereditario. Sin embargo, para la mayoría de las familias, la causa genética de la enfermedad sigue siendo desconocida y se clasifican como cáncer de páncreas familiar (FPC). Los familiares de primer grado sanos de familias con cáncer de páncreas hereditario o FPC son la única población de alto riesgo que se incluye en programas de cribado basados en imágenes para la detección temprana, según las guías europeas e internacionales actuales. Por lo tanto, el objetivo de este estudio fue identificar la base genética del FPC en casos diagnosticados de PDAC con sospecha de FPC, así como la comparación con casos esporádicos. Además, se validaron biomarcadores para la detección temprana de PDAC y sus lesiones precursoras con el objetivo de mejorar el pronóstico. La secuenciación dirigida mediante panel identificó variantes germinales patogénicas y potencialmente patogénicas en genes de cáncer hereditario previamente descritos en el 25% de los casos de FPC, que podrían asociarse con el fenotipo familiar descrito. Estos genes incluyeron MUTYH, ATM, CDKN2A, BRCA2, TP53, CHEK2, CFTR, MLH1, MSH2 y TET2. La naturaleza patogénica de las variantes encontradas se validó mediante un análisis independiente utilizando una plataforma de síndromes de cáncer hereditario basada en la población española (SpadaHC), evaluando su utilidad para estudios futuros. El setenta y cinco por ciento de los casos con antecedentes familiares de PDAC carecían de variantes patogénicas o potencialmente patogénicas en genes de susceptibilidad al cáncer conocidos, aunque se encontraron nuevas variantes de riesgo a través de la secuenciación del exoma completo en la mayoría de estos casos. Estas variantes afectaron a genes proto-oncogénicos (BAG5, PLAU, TNFAIP8, THPO, ACVR1, EIF4EBP1), reparación del ADN (NPR2, CCAR2), genes supresores de tumores (TSPAN32, ING1, IFIH1), componentes del citoesqueleto (KIAA0586, ODF2), estructuras de adhesión (ITGB4, SGCA, FAM187A, DSC3, MUC4, MUC6, MUC16) y transportadores de membrana (ABCA4, ABCB7, ATP1B). Se encontraron variantes patogénicas y potencialmente patogénicas en genes de susceptibilidad al cáncer en casos clasificados clínicamente como esporádicos (ATM, MUTYH, MLH3, KIF1B y CFTR), lo que abre la puerta a futuros estudios para investigar las características de este subconjunto de pacientes. Aproximadamente el 50% de la cohorte de alto riesgo tenía variantes germinales patogénicas y probablemente patogénicas en genes de susceptibilidad al cáncer, en genes como ATM, MLH1, CDKN2A, BRCA1, BRCA2, MUTYH y CFTR. Además, se encontraron variantes adicionales en genes previamente no descritos que podrían asociarse con el fenotipo familiar, particularmente en individuos con anomalías pancreáticas detectadas por pruebas de imagen. Cuatro familias que fueron negativas para variantes patogénicas mediante la secuenciación de paneles fueron analizadas en profundidad mediante secuenciación del exoma. Se encontraron variantes patogénicas y potencialmente patogénicas en los genes CDC45, GLB1, ABCA4, C6, AGER, MTHFR, GJB2, MUC6, MUC4, TAS2R14, CLN3, KISS1R, BCAT1, CPT2 y CCDC141, demostrando la amplia variabilidad genética de este síndrome y la utilidad de los estudios genéticos para identificar genes asociados con el fenotipo familiar. En cuanto a los loci de alto riesgo, se encontró una asociación entre el SNP rs708224 en BICD1 previamente descrito y los casos de PDAC e individuos con antecedentes familiares de PDAC. También se encontró una posible asociación entre el polimorfismo rs7771466 en DPP6 y los casos con FPC, sin embargo, no fue estadísticamente significativa debido al tamaño de la cohorte. Finalmente, se estudiaron diferentes biomarcadores de proteínas basadas en suero para explorar su uso potencial en la detección temprana de PDAC. Se evaluaron los niveles de osteopontina y factor de crecimiento de hepatocitos en suero como biomarcadores discriminativos en diferentes cohortes, incluyendo casos de PDAC, controles sanos e individuos de alto riesgo. Sin embargo, no mejoraron la sensibilidad en comparación con los marcadores estándar actuales, CA19-9 y CEA. Se validó una firma de biomarcadores propuesta para distinguir entre lesiones IPMN de alto y bajo grado, así como entre individuos con y sin anomalías pancreáticas a través de imágenes del páncreas. La combinación de CEA, MUC17 y CSF2RA permitió distinguir a los individuos con lesiones pancreáticas de alto riesgo de aquellos con anomalías de bajo riesgo, con una especificidad del 87.5% y una sensibilidad del 80%. En conclusión, este estudio demostró que los casos de PDAC con antecedentes familiares tienen variantes germinales patogénicas que afectan los procesos de reparación del ADN y la estructura del citoesqueleto que podrían estar relacionadas con el riesgo de PDAC. Se identificaron nuevos genes y variantes en casos de PDAC e individuos de alto riesgo con síndrome de FPC que pueden explicar el fenotipo de la enfermedad en estas familias. La detección de variantes potencialmente patogénicas en casos aparentemente esporádicos sugiere que no solo los antecedentes familiares definen el riesgo. Además, los paneles de biomarcadores basados en suero han permitido la creación de modelos predictivos con una alta sensibilidad y especificidad para identificar a individuos con mayor riesgo de progresión a PDAC

Role of Distal Regulatory Elements in Cancer Progression and Therapy

Enhancer elements comprise of regions of DNA that are distal to gene promoters with a characteristic capacity to affect and regulate gene transcription. Enhancers are enriched in a highly context-specific manner allowing for intricate control of gene expression. Current studies endeavor to elucidate the mechanisms underlying enhancer activation and function to ultimately exploit their specificity in targeted therapeutics. Due to the reported addiction of cancer to aberrant gene transcription, targeting enhancer elements is a promising therapeutic target in various malignancies. In this project, we conduct a series of studies with the general aim of extending the knowledge about the molecular mechanisms by which enhancers drive aberrant transcription in cancer. We focus on epigenetic modulation to exploit enhancer elements as therapeutic or prognostic targets. In the first study included in our project, we evaluated the importance of the super enhancer subcategory of distal regulatory elements in a breast cancer cell line where estrogen plays an important role in driving gene expression through enhancers. Super enhancers are claimed to be a highly active subgroup of distal regulatory elements that is abundantly enriched with transcription factors, span long stretches of DNA, and exhibit preferential efficacy in driving major transcriptional programs in cancer. We identified super enhancers related to estrogen in this system using the standard algorithm and failed to observe a distinct high efficacy of super enhancers compared to typical enhancers. By varying the settings of this algorithm, we also uncovered biases in enhancer identification that extensively influence the results. On the other hand, we observed that major targets of estrogen activation showed a preference for association with super enhancers and concluded that they may indeed tend to regulate the transcription of master regulators. Accordingly, we concluded that the focused attention given to super enhancers should not lead to disregarding typical enhancers which also play a significant and important role in gene transcription regulation. Consequently, in the second study we reviewed the role of enhancers in pancreatic cancer, a malignancy with exceptionally low survival rates. We focused on the application of epigenetic modulators, such as bromodomain and extraterminal proteins inhibitors and histone deactylase inhibitors, in targeting enhancer elements and speculated about mechanisms underlying the reported synergy between these two inhibitors. Interestingly, we used publicly available data to further study the context-specificity of enhancers. Notably, we observed a tendency where the same oncogenic target gene is activated by different enhancers in various systems due to differential expression of transcription factors. Accordingly, we expanded our studies in pancreatic cancer and uncovered a group of subtype-specific super enhancers that drive the cells into a squamous phenotype which correlates with a particularly poor prognosis. Studying the general activation epigenetic profiles of different pancreatic cancer cell lines identified deltaNp63 as a major driver of the squamous molecular identity in cells and patient-derived xenografts. Moreover, extensive analysis of the role of deltaNp63 in driving a more aggressive phenotype uncovered the implication of super enhancers which are supported by a network of interconnected and differentially expressed transcription factors. This pattern resembles the reports of transcription factor regulatory circuitry driving the pluripotent molecular identity of embryonic stem cells. Identification of the same pattern governing differentiation into specific molecular subtypes in pancreatic cancer opens the door to precision-based medicine approaches targeting this circuitry in this particular subtype. Finally, we further investigated the role of enhancer elements in the context of chemotherapeutic resistance in pancreatic cancer. Interestingly, we observed that pro-inflammatory and migratory programs are activated in paclitaxel-resistant cells via activation of BET-dependent enhancers. Furthermore, we observed that BET inhibition sensitizes resistant and sensitive cells to paclitaxel. Notably, super enhancers that we observed to be enriched in resistant cells were associated with genes that correlate with poor prognosis. This study confirmed the patterns we uncovered in the other studies where enhancers and super enhancers drive aberrant transcription activation in cancer and present a promising target for patient treatment. Altogether, this project resulted in 2 peer-reviewed publications in the journals of Transcription and Epigenomes, one manuscript that has been peer-reviewed and is currently under revision in Proceedings of the National Academy of Sciences of the United States of America (PNAS), and another manuscript in preparation for submission. These publications/manuscripts join the growing body of literature investigating the role of enhancers in malignancy and aim to guide new approaches for precision-based medicine.2019-12-1

The Role of Long Noncoding RNAs in Cancer and Microbiota-derived Aryl Hydrocarbon Receptor Ligands

LncRNAs are a group of non-coding RNAs containing >200 nucleotides and these RNAs have no significant protein coding potential. In the past 10-15 years the role of lncRNAs in cancers have been demonstrated, however, their function in tumors and potential for drug targeting are not well defined. We studied two lncRNAs, HOXA transcript at the distal tip (HOTTIP) and metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in pancreatic cancer using the in vitro cell lines and in vivo xenograft or transgenic mouse models. Our results demonstrated that both lncRNAs are pro-oncogenic in the pancreatic cancer, supported by the observation that knockdown of HOTTIP and MALAT1 decreased cell proliferation, migration/invasion and increased apoptosis. HOTTIP might exhibit some pro-oncogenic functions, via the regulation of HOXA gene clusters in a cis-regulating manner. On the other hand, MALAT1 regulate responses of pancreatic cancer cells in part via polycomb repressive complex 2 (PRC2) dependent and independent pathways. Our transcriptomic results support the important but distinct roles of HOTTIP and MALAT1 in pancreatic cancer and we also show that MALAT1 expression can be targeted by small molecule drugs. In the second part of this dissertation, we studied several microbiota-derived aryl hydrocarbon receptor (AhR) ligands. The AhR is a ligand-activated transcription factor with an evolving role in the normal physiological development and diseases. Gut microbiota metabolites are important for mediating communication between gut microflora and the host. It has recently been shown that the gut microbiota produces several metabolites that are AhR ligands. Microbiota-derived tryptophan metabolites and 1,4-dihydroxy-2-napthoic acid (DHNA) and related compounds are reported to be AhR ligands as evidence by their induction of cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) and other AhR-responsive genes. We hypothesized the microbiota-derived AhR ligands are selective AhR modulators (sAhRMs) and their induction responses are compound, gene and cell context dependent. We have carried out extensive studies on tryptophan metabolites, DHNA and related compounds in both human and mouse colon cancer cell lines, and have observed that some of these compounds exhibited partial agonist/antagonist activities that are both gene and cell context specific

The Role of Inter-Alpha-Trypsin-Inhibitor-Heavy-Chain-5 (ITIH5) in Suppressing Pancreatic Cancer Metastasis

Metastasis is the spread of cancer from the site of origin to a distant site, where the colony of malignant cells grows. Growth of malignant cells in secondary sites disrupts organ function and increases the tumor burden on the host. Thus, there is a critical need to understand the factors regulating the spread of cancer cells to distant sites and their growth therein. Over half of all patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) present with liver metastasis, and five-year survival for these patients is currently three percent. We performed an in vivo screen for PDAC metastasis suppressors using a human whole-genome shRNA library in order to understand factors regulating PDAC metastasis and identified Inter-α-Trypsin Inhibitor Heavy Chain 5 (ITIH5) as a suppressor of PDAC metastasis. Knockdown of ITIH5 significantly increased liver metastasis while high ITIH5 expression was correlated with rounded cell morphology and decreased cell motility and metastasis. ITIH5 is a secreted protein related to plasma protease inhibitors. To test the hypothesis that secretion of ITIH5 is required to suppress metastasis, we deleted the secretion signal sequence of ITIH5 (ITIH5Δs) and compared development of liver metastasis in highly metastatic PDAC cells expressing either control vector, secreted ITIH5, or secretion-deficient ITIH5Δs. Intriguingly, ITIH5Δs was sufficient to recapitulate the effects of secreted ITIH5 on cell morphology, motility and metastasis. These data suggest that ITIH5 may suppress metastasis by an intracellular mechanism not predicted by previously published reports. Understanding how intracellular ITIH5 attenuates PDAC metastasis could reveal new elements of PDAC biology that could become future therapeutic targets for patients affected by this aggressive cancer