Vassiliki Karantza-Wadsworth

Role of autophagy in cancer

Autophagy is a cellular degradation pathway for the clearance of damaged or superfluous proteins and organelles. The recycling of these intracellular constituents also serves as an alternative energy source during periods of metabolic stress to maintain homeostasis and viability. In tumour cells with defects in apoptosis, autophagy allows prolonged survival. Paradoxically, autophagy defects are associated with increased tumorigenesis, but the mechanism behind this has not been determined. Recent evidence suggests that autophagy provides a protective function to limit tumour necrosis and inflammation, and to mitigate genome damage in tumour cells in response to metabolic stress.

Autophagy Suppresses Tumorigenesis through Elimination of p62

Allelic loss of the essential autophagy gene beclin1 occurs in human cancers and renders mice tumor-prone suggesting that autophagy is a tumor-suppression mechanism. While tumor cells utilize autophagy to survive metabolic stress, autophagy also mitigates the resulting cellular damage that may limit tumorigenesis. In response to stress, autophagy-defective tumor cells preferentially accumulated p62/SQSTM1 (p62), endoplasmic reticulum (ER) chaperones, damaged mitochondria, reactive oxygen species (ROS), and genome damage. Moreover, suppressing ROS or p62 accumulation prevented damage resulting from autophagy defects indicating that failure to regulate p62 caused oxidative stress. Importantly, sustained p62 expression resulting from autophagy defects was sufficient to alter NF-kappaB regulation and gene expression and to promote tumorigenesis. Thus, defective autophagy is a mechanism for p62 upregulation commonly observed in human tumors that contributes directly to tumorigenesis likely by perturbing the signal transduction adaptor function of p62-controlling pathways critical for oncogenesis.

Role and regulation of autophagy in cancer

Autophagy is an evolutionarily conserved process whereby cytoplasm and cellular organelles are degraded in lysosomes for amino acid and energy recycling. Autophagy is a survival pathway activated in response to nutrient deprivation and other stressful stimuli, such as metabolic stress and exposure to anticancer drugs. However, autophagy may also result in cell death, if it proceeds to completion. Defective autophagy is implicated in tumorigenesis, as the essential autophagy regulator beclin 1 is monoallelically deleted in human breast, ovarian and prostate cancers, and beclin 1(+/-) mice are tumor-prone. How autophagy suppresses tumorigenesis is under intense investigation. Cell-autonomous mechanisms, involving protection of genome integrity and stability, and a non-cell-autonomous mechanism, involving suppression of necrosis and inflammation, have been discovered so far. The role of autophagy in treatment responsiveness is also complex. Autophagy inhibition concurrently with chemotherapy or radiotherapy has emerged as a novel approach in cancer treatment, as autophagy-competent tumor cells depend on autophagy for survival under drug- and radiation-induced stress. Alternatively, autophagy stimulation and preservation of cellular fitness by maintenance of protein and organelle quality control, suppression of DNA damage and genomic instability, and limitation of necrosis-associated inflammation may play a critical role in cancer prevention.

Role of the Polarity Determinant Crumbs in Suppressing Mammalian Epithelial Tumor Progression

Most tumors are epithelial-derived, and although disruption of polarity and aberrant cellular junction formation is a poor prognosticator in human cancer, the role of polarity determinants in oncogenesis is poorly understood. Using in vivo selection, we identified a mammalian orthologue of the Drosophila polarity regulator crumbs as a gene whose loss of expression promotes tumor progression. Immortal baby mouse kidney epithelial cells selected in vivo to acquire tumorigenicity displayed dramatic repression of crumbs3 (crb3) expression associated with disruption of tight junction formation, apicobasal polarity, and contact-inhibited growth. Restoration of crb3 expression restored junctions, polarity, and contact inhibition while suppressing migration and metastasis. These findings suggest a role for mammalian polarity determinants in suppressing tumorigenesis that may be analogous to the well-studied polarity tumor suppressor mechanisms in Drosophila.

Bcl-2 Modulation to Activate Apoptosis in Prostate Cancer

Apoptosis resistance is a hallmark of cancer linked to disease progression and treatment resistance, which has led to the development of anticancer therapeutics that restore apoptotic function. Antiapoptotic Bcl-2 is frequently overexpressed in refractory prostate cancer and increased following standard hormonal therapy and chemotherapy; however, the rationally designed Bcl-2 antagonist, ABT-737, has not shown single agent apoptosis-promoting activity against human prostate cancer cell lines. This is likely due to the coordinate expression of antiapoptotic, Bcl-2–related Mcl-1 that is not targeted by ABT-737. We developed a mouse model for prostate cancer in which apoptosis resistance and tumorigenesis were conferred by Bcl-2 expression. Combining ABT-737 with agents that target Mcl-1 sensitized prostate cancer cell lines with an apoptotic block to cell death in vitro. In mice in vivo, ABT-737 showed single agent efficacy in prostate tumor allografts in which tumor cells are under hypoxic stress. In human prostate cancer tissue, examined using a novel tumor explant system designated Tumor Tissue Assessment for Response to Chemotherapy, combination chemotherapy promoted efficient apoptosis. Thus, rational targeting of both the Bcl-2 and Mcl-1 mechanisms of apoptosis resistance may be therapeutically advantageous for advanced prostate cancer. (Mol Cancer Res 2009;7(9):1487–96)

Assessing metabolic stress and autophagy status in epithelial tumors.

Autophagy is a survival mechanism activated in response to metabolic stress. In normal tissues autophagy plays a major role in energy homeostasis through catabolic self-digestion of damaged proteins and organelles. Contrary to its survival function, autophagy defects are implicated in tumorigenesis suggesting that autophagy is a tumor suppression mechanism. Although the exact mechanism of this tumor suppressor function is not known, it likely involves mitigation of cellular damage leading to chromosomal instability. The complex role of functional autophagy in tumors calls for model systems that allow the assessment of autophagy status, stress management and the impact on oncogenesis both in vitro as well as in vivo. We developed model systems that involve generation of genetically defined, isogenic and immortal epithelial cells from different tissue types that are applicable to both wild-type and mutant mice. This permits the study of tissue- as well as gene-specific tumor promoting functions. We successfully employed this strategy to generate isogenic, immortal epithelial cell lines from wild-type and mutant mice deficient in essential autophagy genes such as beclin 1 (beclin 1(+/-)) and atg5 (atg 5(-/-)). As these cell lines are amenable to further genetic manipulation, they allowed us to generate cell lines with apoptosis defects and stable expression of the autophagy marker EGFP-LC3 that facilitate in vitro and in vivo assessment of stress-mediated autophagy induction. We applied this model system to directly monitor autophagy in cells and 3D-morphogenesis in vitro as well as in tumor allografts in vivo. Using this model system we demonstrated that autophagy is a survival response in solid tumors that co-localizes with hypoxic regions, allowing tolerance to metabolic stress. Furthermore, our studies have established that autophagy also protects tumor cells from genome damage and limits cell death and inflammation as possible means to tumor suppression. Additionally these cell lines provide an efficient way to perform biochemical analyses, and high throughput screening for modulators of autophagy for potential use in cancer therapy and prevention.

A Mouse Mammary Epithelial Cell Model to Identify Molecular Mechanisms Regulating Breast Cancer Progression

Breast cancer, like any other human cancer, results from the accumulation of mutations that deregulate critical cellular processes, such as cell proliferation and death. Activation of oncogenes and inactivation of tumor suppressor genes are common events during cancer initiation and progression and often determine treatment responsiveness. Thus, recapitulating these events in mouse cancer models is critical for unraveling the molecular mechanisms involved in tumorigenesis and for interrogating their possible impact on response to anticancer drugs. We have developed a novel mouse mammary epithelial cell model, which replicates the steps of epithelial tumor progression and takes advantage of the power of mouse genetics and the ability to assess three-dimensional morphogenesis in the presence of extracellular matrix to model human breast cancer.

Abstract CN05-02: Autophagy and mammary tumorigenesis: role of metabolic and ER stress management

Abstracts: Frontiers in Cancer Prevention Research 2008 CN05-02 Autophagy is an evolutionarily conserved catabolic process whereby cellular organelles and bulk cytoplasm are targeted to lysosomes for degradation. Autophagy is a survival pathway required for cell viability during starvation; however, if it proceeds to completion, autophagy can lead to cell death. Autophagy also plays a role in tumorigenesis, as the essential autophagy regulator beclin 1 is monoallelically deleted in many human ovarian, breast, and prostate cancers, and beclin 1+/- mice are tumor-prone. Thus, autophagy likely suppresses tumorigenesis, but the mechanism has been unknown. We recently showed that allelic loss of beclin 1 compromises the autophagy potential of immortalized mouse mammary epithelial cells (iMMECs) in vitro and in mammary tumors in vivo, sensitizes iMMECs to metabolic stress and accelerates lumen formation in mammary acini. Autophagy defects also activate the DNA damage response in vitro and in mammary tumors in vivo, promote gene amplification, and synergize with defective apoptosis to accelerate mammary tumorigenesis. We investigated the mechanism by which autophagy mitigates metabolic stress and limits genome damage in mammary epithelial cells, so as to gain insight into how autophagy suppresses mammary tumorigenesis. A proteomic analysis was performed to determine how autophagy-competent mammary epithelial cells respond successfully to metabolic stress as compared to cells with autophagy defects. Protein extracts from apoptosis-defective beclin 1+/+ and beclin 1+/- iMMECs exposed to metabolic stress for 0, 4 and 7 days were analyzed by two-dimensional difference gel electrophoresis (2D-DIGE). More than a hundred proteins with differential expression under metabolic stress were sequenced and mostly represented members of the GRP chaperone system, cellular metabolism, mitochondrial and cytoskeletal proteins. The 2D-DIGE results were validated for a subset of the sequenced proteins on beclin 1+/+ and beclin 1+/- iMMECs under metabolic stress in vitro and on beclin 1+/+ and beclin 1+/- iMMEC-generated mammary tumors in vivo. Expression of several proteins upregulated under metabolic stress was also examined in 3D-morphogenesis assays. For most proteins, expression was higher in the central acinar cells, indicating that the acinar center is a physiologic area of increased metabolic stress. Differential protein expression between wild-type and autophagy-defective iMMECs under conditions of metabolic stress is currently being validated on mammary tissues from beclin 1+/+ and beclin 1+/- female mice. Therefore, failure of autophagy-deficient mammary tumor cells to effectively manage metabolic stress is associated with increased unfolded protein load and endoplasmic reticulum (ER) stress, which may in turn play a pivotal role in promoting genome damage and instability, and thus cancer progression, especially when apoptosis is concurrently inactivated. These studies identify new therapeutic targets and provide valuable clues into how to best modulate autophagy for cancer prevention and treatment. Citation Information: Cancer Prev Res 2008;1(7 Suppl):CN05-02.