Autophagy is required for mammary tumor recurrence by promoting dormant tumor cell survival following therapy.

BACKGROUND: Mortality from breast cancer is principally due to tumor recurrence. Recurrent breast cancers arise from the pool of residual tumor cells, termed minimal residual disease, that survive treatment and may exist in a dormant state for 20 years or more following treatment of the primary tumor. As recurrent breast cancer is typically incurable, understanding the mechanisms underlying dormant tumor cell survival is a critical priority in breast cancer research. The importance of this goal is further underscored by emerging evidence suggesting that targeting dormant residual tumor cells in early-stage breast cancer patients may be a means to prevent tumor recurrence and its associated mortality. In this regard, the role of autophagy in dormant tumor cell survival and recurrence remains unresolved, with conflicting reports of both pro-survival/recurrence-promoting and pro-death/recurrence-suppressing effects of autophagy inhibition in dormant tumor cells. Resolving this question has important clinical implications. METHODS: We used genetically engineered mouse models that faithfully recapitulate key features of human breast cancer progression, including minimal residual disease, tumor dormancy, and recurrence. We used genetic and pharmacological approaches to inhibit autophagy, including treatment with chloroquine, genetic knockdown of ATG5 or ATG7, or deletion of BECN and determined their effects on dormant tumor cell survival and recurrence. RESULTS: We demonstrate that the survival and recurrence of dormant mammary tumor cells following therapy is dependent upon autophagy. We find that autophagy is induced in vivo following HER2 downregulation and remains activated in dormant residual tumor cells. Using genetic and pharmacological approaches we show that inhibiting autophagy by chloroquine administration, ATG5 or ATG7 knockdown, or deletion of a single allele of the tumor suppressor Beclin 1 is sufficient to inhibit mammary tumor recurrence, and that autophagy inhibition results in the death of dormant mammary tumor cells in vivo. CONCLUSIONS: Our findings demonstrate a pro-tumorigenic role for autophagy in tumor dormancy and recurrence following therapy, reveal that dormant tumor cells are uniquely reliant upon autophagy for their survival, and indicate that targeting dormant residual tumor cells by inhibiting autophagy impairs tumor recurrence. These studies identify a pharmacological target for a cellular state that is resistant to commonly used anti-neoplastic agents and suggest autophagy inhibition as an approach to reduce dormant minimal residual disease in order to prevent lethal tumor recurrence.

Cellular dormancy in minimal residual disease following targeted therapy.

BACKGROUND: Breast cancer mortality is principally due to tumor recurrence, which can occur following extended periods of clinical remission that may last decades. While clinical latency has been postulated to reflect the ability of residual tumor cells to persist in a dormant state, this hypothesis remains unproven since little is known about the biology of these cells. Consequently, defining the properties of residual tumor cells is an essential goal with important clinical implications for preventing recurrence and improving cancer outcomes. METHODS: To identify conserved features of residual tumor cells, we modeled minimal residual disease using inducible transgenic mouse models for HER2/neu and Wnt1-driven tumorigenesis that recapitulate cardinal features of human breast cancer progression, as well as human breast cancer cell xenografts subjected to targeted therapy. Fluorescence-activated cell sorting was used to isolate tumor cells from primary tumors, residual lesions following oncogene blockade, and recurrent tumors to analyze gene expression signatures and evaluate tumor-initiating cell properties. RESULTS: We demonstrate that residual tumor cells surviving oncogenic pathway inhibition at both local and distant sites exist in a state of cellular dormancy, despite adequate vascularization and the absence of adaptive immunity, and retain the ability to re-enter the cell cycle and give rise to recurrent tumors after extended latency periods. Compared to primary or recurrent tumor cells, dormant residual tumor cells possess unique features that are conserved across mouse models for human breast cancer driven by different oncogenes, and express a gene signature that is strongly associated with recurrence-free survival in breast cancer patients and similar to that of tumor cells in which dormancy is induced by the microenvironment. Although residual tumor cells in both the HER2/neu and Wnt1 models are enriched for phenotypic features associated with tumor-initiating cells, limiting dilution experiments revealed that residual tumor cells are not enriched for cells capable of giving rise to primary tumors, but are enriched for cells capable of giving rise to recurrent tumors, suggesting that tumor-initiating populations underlying primary tumorigenesis may be distinct from those that give rise to recurrence following therapy. CONCLUSIONS: Residual cancer cells surviving targeted therapy reside in a well-vascularized, desmoplastic microenvironment at both local and distant sites. These cells exist in a state of cellular dormancy that bears little resemblance to primary or recurrent tumor cells, but shares similarities with cells in which dormancy is induced by microenvironmental cues. Our observations suggest that dormancy may be a conserved response to targeted therapy independent of the oncogenic pathway inhibited or properties of the primary tumor, that the mechanisms underlying dormancy at local and distant sites may be related, and that the dormant state represents a potential therapeutic target for preventing cancer recurrence.

Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer.

Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitt's Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors.

Inhibition of the single downstream target BAG1 activates the latent apoptotic potential of MYC.

Aberrant MYC expression is a common oncogenic event in human cancer. Paradoxically, MYC can either drive cell cycle progression or induce apoptosis. The latent ability of MYC to induce apoptosis has been termed "intrinsic tumor suppressor activity," and reactivating this apoptotic function in tumors is widely considered a valuable therapeutic goal. As a transcription factor, MYC controls the expression of many downstream targets, and for the majority of these, it remains unclear whether or not they play direct roles in MYC function. To identify the subset of genes specifically required for biological activity, we conducted a screen for functionally important MYC targets and identified BAG1, which encodes a prosurvival chaperone protein. Expression of BAG1 is regulated by MYC in both a mouse model of breast cancer and transformed human cells. Remarkably, BAG1 induction is essential for protecting cells from MYC-induced apoptosis. Ultimately, the synthetic lethality we have identified between MYC overexpression and BAG1 inhibition establishes a new pathway that might be exploited to reactivate the latent apoptotic potential of MYC as a cancer therapy.