Faculty Bibliography

Pancreatic ductal adenocarcinoma (PDAC) is amongst the deadliest of human cancers, due to its late diagnosis as well as its intense resistance to currently available therapeutics. To identify mechanisms as to why PDAC are refractory to DNA damaging cytoxic chemotherapy and radiation, we performed a global interrogation of the DNA damage response of PDAC. We find that PDAC cells generally harbor high levels of spontaneous DNA damage. Inhibition of Non-Homologous End Joining (NHEJ) repair either pharmacologically or by RNAi resulted in a further accumulation of DNA damage, inhibition of growth, and ultimately apoptosis even in the absence of exogenous DNA damaging agents. In response to radiation, PDAC cells rely on the NHEJ pathway to rapidly repair DNA double strand breaks. Mechanistically, when NHEJ is inhibited there is a compensatory increase in Homologous Recombination (HR). Despite this upregulation of HR, DNA damage persists and cells are significantly more sensitive to radiation. Together, these findings support the incorporation of NHEJ inhibition into PDAC therapeutic approaches, either alone, or in combination with DNA damaging therapies such as radiation.

Autophagy inhibition is a novel cancer therapeutic strategy in the early stages of clinical trial testing. The initial rationale for using autophagy inhibition was generated by research revealing that autophagy is upregulated in response to external stresses, including chemotherapy and radiotherapy. Combining autophagy inhibition with agents that induce autophagy as a pro-survival response may therefore increase their therapeutic efficacy. Recent research has shown that some cancer cells, particularly those driven by the K-Ras oncogene, also depend on elevated levels of autophagy for survival even in the absence of external stressors. In multiple in vitro as well as in vivo systems, oncogenic Ras-mediated transformation and tumor growth are dependent on autophagy to evade metabolic stress and cell death. These studies have subsequently led to further early phase clinical testing whether autophagy inhibition is a viable and effective strategy for targeting Ras-driven tumors. Even before the clinical results are available from these ongoing clinical trials, much work remains to optimally develop the approach of autophagy inhibition clinically; most notably reliably detecting levels of autophagy in human tumor samples, pharmacodynamics of currently available autophagy inhibitors (chloroquine and the derivative hydroxychloroquine), and new target identification and drug development.

Macroautophagy (referred to hereafter as autophagy) is a highly regulated cellular process that serves to remove damaged proteins and organelles from the cell. Autophagy contributes to an array of normal and pathological processes, and has recently emerged as a key regulator of multiple aspects of cancer biology. The role of autophagy in cancer is complex and is likely dependent on tumor type, stage, and genetic context. This complexity is illustrated by the identification of settings where autophagy acts potently to either promote or inhibit tumorigenesis. In this review, I discuss the underlying basis for these opposing functions and propose a model suggesting a dynamic role for autophagy in malignancy. Collectively, the data point to autophagy as serving as a barrier to limit tumor initiation. Once neoplastic lesions are established, it appears that adaptive changes occur that now result in positive roles for autophagy in malignant progression and in subsequent tumor maintenance. Remarkably, constitutive activation of autophagy is critical for continued growth of some tumors, serving to both reduce oxidative stress and provide key intermediates to sustain cell metabolism. Autophagy is also induced in response to cancer therapies where it can function as a survival mechanism that limits drug efficacy. These findings have inspired significant interest in applying anti-autophagy therapies as an entirely new approach to cancer treatment. It is now apparent that aberrant control of autophagy is among the key hallmarks of cancer. While much needs to be learned about the regulation and context-dependent biological functions of autophagy, it seems clear that modulation of this process will be an attractive avenue for future cancer therapeutic approaches.

Autophagy is a regulated catabolic process that leads to the lysosomal degradation of damaged proteins, organelles and other macromolecules, with subsequent recycling of bioenergetic intermediates. The role of autophagy in cancer is undoubtedly complex and likely dependent on tumor type and on the cellular and developmental context. While it has been well demonstrated that autophagy may function as a tumor suppressor, there is mounting evidence that autophagy may have pro-tumorigenic roles, e.g., promoting therapeutic resistance as well as survival under stresses such as hypoxia and nutrient deprivation. These two, seemingly disparate functions can be reconciled by a possible temporal role of autophagy during tumor development, initially suppressing tumor initiation yet supporting tumor growth at later stages.

Initiation of pancreatic ductal adenocarcinoma (PDAC) is driven by oncogenic KRAS mutation, and disease progression is associated with frequent loss of tumor suppressors. In this study, human PDAC genome analyses revealed frequent deletion of the PTEN gene as well as loss of expression in primary tumor specimens. A potential role for PTEN as a haploinsufficient tumor suppressor is further supported by mouse genetic studies. The mouse PDAC driven by oncogenic Kras mutation and Pten deficiency also sustains spontaneous extinction of Ink4a expression and shows prometastatic capacity. Unbiased transcriptomic analyses established that combined oncogenic Kras and Pten loss promotes marked NF-kappaB activation and its cytokine network, with accompanying robust stromal activation and immune cell infiltration with known tumor-promoting properties. Thus, PTEN/phosphoinositide 3-kinase (PI3K) pathway alteration is a common event in PDAC development and functions in part to strongly activate the NF-kappaB network, which may serve to shape the PDAC tumor microenvironment.

Macroautophagy (autophagy) is a regulated catabolic pathway to degrade cellular organelles and macromolecules. The role of autophagy in cancer is complex and may differ depending on tumor type or context. Here we show that pancreatic cancers have a distinct dependence on autophagy. Pancreatic cancer primary tumors and cell lines show elevated autophagy under basal conditions. Genetic or pharmacologic inhibition of autophagy leads to increased reactive oxygen species, elevated DNA damage, and a metabolic defect leading to decreased mitochondrial oxidative phosphorylation. Together, these ultimately result in significant growth suppression of pancreatic cancer cells in vitro. Most importantly, inhibition of autophagy by genetic means or chloroquine treatment leads to robust tumor regression and prolonged survival in pancreatic cancer xenografts and genetic mouse models. These results suggest that, unlike in other cancers where autophagy inhibition may synergize with chemotherapy or targeted agents by preventing the up-regulation of autophagy as a reactive survival mechanism, autophagy is actually required for tumorigenic growth of pancreatic cancers de novo, and drugs that inactivate this process may have a unique clinical utility in treating pancreatic cancers and other malignancies with a similar dependence on autophagy. As chloroquine and its derivatives are potent inhibitors of autophagy and have been used safely in human patients for decades for a variety of purposes, these results are immediately translatable to the treatment of pancreatic cancer patients, and provide a much needed, novel vantage point of attack.

Pancreas ductal adenocarcinoma (PDAC) is a highly lethal cancer that typically presents as advanced, unresectable disease. This invasive tendency, coupled with intrinsic resistance to standard therapies and genome instability, are major contributors to poor long-term survival. The genetic elements governing the invasive propensity of PDAC have not been well elucidated. Here, in the course of validating resident genes in highly recurrent and focal amplifications in PDAC, we have identified Rio Kinase 3 (RIOK3) as an amplified gene that alters cytoskeletal architecture as well as promotes pancreatic ductal cell migration and invasion. We determined that RIOK3 promotes its invasive activities through activation of the small G protein, Rac. This genomic and functional link to Rac signaling prompted a genome wide survey of other components of the Rho family network, revealing p21 Activated Kinase 4 (PAK4) as another amplified gene in PDAC tumors and cell lines. Like RIOK3, PAK4 promotes pancreas ductal cell motility and invasion. Together, the genomic and functional profiles establish the Rho family GTP-binding proteins as integral to the hallmark invasive nature of this lethal disease.

Glioblastoma (GBM) is a highly lethal brain tumour presenting as one of two subtypes with distinct clinical histories and molecular profiles. The primary GBM subtype presents acutely as a high-grade disease that typically harbours mutations in EGFR, PTEN and INK4A/ARF (also known as CDKN2A), and the secondary GBM subtype evolves from the slow progression of a low-grade disease that classically possesses PDGF and TP53 events. Here we show that concomitant central nervous system (CNS)-specific deletion of p53 and Pten in the mouse CNS generates a penetrant acute-onset high-grade malignant glioma phenotype with notable clinical, pathological and molecular resemblance to primary GBM in humans. This genetic observation prompted TP53 and PTEN mutational analysis in human primary GBM, demonstrating unexpectedly frequent inactivating mutations of TP53 as well as the expected PTEN mutations. Integrated transcriptomic profiling, in silico promoter analysis and functional studies of murine neural stem cells (NSCs) established that dual, but not singular, inactivation of p53 and Pten promotes an undifferentiated state with high renewal potential and drives increased Myc protein levels and its associated signature. Functional studies validated increased Myc activity as a potent contributor to the impaired differentiation and enhanced renewal of NSCs doubly null for p53 and Pten (p53(-/-) Pten(-/-)) as well as tumour neurospheres (TNSs) derived from this model. Myc also serves to maintain robust tumorigenic potential of p53(-/-) Pten(-/-) TNSs. These murine modelling studies, together with confirmatory transcriptomic/promoter studies in human primary GBM, validate a pathogenetic role of a common tumour suppressor mutation profile in human primary GBM and establish Myc as an important target for cooperative actions of p53 and Pten in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal and tumorigenic potential.

Glioblastoma (GBM) is a highly lethal primary brain cancer with hallmark features of diffuse invasion, intense apoptosis resistance and florid necrosis, robust angiogenesis, and an immature profile with developmental plasticity. In the course of assessing the developmental consequences of central nervous system (CNS)-specific deletion of p53 and Pten, we observed a penetrant acute-onset malignant glioma phenotype with striking clinical, pathological, and molecular resemblance to primary GBM in humans. This primary, as opposed to secondary, GBM presentation in the mouse prompted genetic analysis of human primary GBM samples that revealed combined p53 and Pten mutations as the most common tumor suppressor defects in primary GBM. On the mechanistic level, the "multiforme" histopathological presentation and immature differentiation marker profile of the murine tumors motivated transcriptomic promoter-binding element and functional studies of neural stem cells (NSCs), which revealed that dual, but not singular, inactivation of p53 and Pten promotes cellular c-Myc activation. This increased c-Myc activity is associated not only with impaired differentiation, enhanced self-renewal capacity of NSCs, and tumor-initiating cells (TICs), but also with maintenance of TIC tumorigenic potential. Together, these murine studies have provided a highly faithful model of primary GBM, revealed a common tumor suppressor mutational pattern in human disease, and established c-Myc as a key component of p53 and Pten cooperative actions in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal, and tumorigenic potential.

Colorectal cancer (CRC) is a major cause of cancer morbidity and mortality, and elucidation of its underlying genetics has advanced diagnostic screening, early detection, and treatment. Because CRC genomes are characterized by numerous non-random chromosomal structural alterations, we sought to delimit regions of recurrent amplifications and deletions in a collection of 42 primary specimens and 37 tumor cell lines derived from chromosomal instability neoplasia and microsatellite instability neoplasia CRC subtypes and to compare the pattern of genomic aberrations in CRC with those in other cancers. Application of oligomer-based array-comparative genome hybridization and custom analytic tools identified 50 minimal common regions (MCRs) of copy number alterations, 28 amplifications, and 22 deletions. Fifteen were highly recurrent and focal (<12 genes) MCRs, five of them harboring known CRC genes including EGFR and MYC with the remaining 10 containing a total of 65 resident genes with established links to cancer. Furthermore, comparisons of these delimited genomic profiles revealed that 22 of the 50 CRC MCRs are also present in lung cancer, glioblastoma, and/or multiple myeloma. Among 22 shared MCRs, nine do not contain genes previously shown genetically altered in cancer, whereas the remaining 13 harbor 35 known cancer genes, of which only 14 have been linked to CRC pathogenesis. Together, these observations point to the existence of many yet-to-be discovered cancer genes driving CRC development, as well as other human cancers, and show the utility of high-resolution copy number analysis in the identification of genetic events common and specific to the development of various tumor types.