NBR1-dependent selective autophagy is required for efficient cell-matrix adhesion site disassembly

Macroautophagy/autophagy has classically been recognized for its vital role in supporting cellular survival during various stresses. However, emerging work has demonstrated that selective autophagy has an impact on diverse cell biological processes by mediating the degradation of various cellular contents during normal cellular homeostasis. We recently established that selective autophagy supports cell migration by promoting the turnover of integrin-based cell-matrix adhesion sites, or focal adhesions (FAs). The autophagy cargo receptor NBR1 acts as a critical mediator of this pathway by promoting targeting of autophagosomes to FAs, leading to their disassembly via the sequestration of FA proteins. Our results demonstrate FAs as a new cellular target for selective autophagy

NBR1-dependent selective autophagy is required for efficient cell-matrix adhesion site disassembly

Macroautophagy/autophagy has classically been recognized for its vital role in supporting cellular survival during various stresses. However, emerging work has demonstrated that selective autophagy has an impact on diverse cell biological processes by mediating the degradation of various cellular contents during normal cellular homeostasis. We recently established that selective autophagy supports cell migration by promoting the turnover of integrin-based cell-matrix adhesion sites, or focal adhesions (FAs). The autophagy cargo receptor NBR1 acts as a critical mediator of this pathway by promoting targeting of autophagosomes to FAs, leading to their disassembly via the sequestration of FA proteins. Our results demonstrate FAs as a new cellular target for selective autophagy

Cellular and metabolic functions for autophagy in cancer cells

Autophagy is a lysosomal degradation pathway that acts as a dynamic regulator of tumorigenesis. Specifically, autophagy has been shown to impede early cancer development while facilitating advanced tumor progression. Recent studies have uncovered several tumor-promoting functions for autophagy; these include the maintenance of multiple metabolic pathways critical for aggressive tumor growth and the promotion of tumor cell survival downstream of the unfolded protein response. Furthermore, autophagy supports anoikis resistance and cancer cell invasion. At the same time, because autophagy cargo receptors, which are essential for selective autophagy, lie upstream of diverse cancer-promoting signaling pathways, they may profoundly influence how alterations in autophagy affect tumor development. This review focuses on how these tumor cell autonomous functions of autophagy broadly impact tumorigenesis

Autophagy-Dependent Production of Secreted Factors Facilitates Oncogenic RAS-Driven Invasion

The tumor promoting functions of autophagy are primarily attributed to its ability to promote cancer cell survival. However, emerging evidence suggests that autophagy plays other roles during tumorigenesis. Here, we uncover that autophagy promotes oncogenic RAS-driven invasion. In epithelial cells transformed with oncogenic RAS, depletion of autophagy-related genes suppresses invasion in three-dimensional culture, decreases cell motility, and reduces pulmonary metastases in vivo. Treatment with conditioned media from autophagy-competent cells rescues the invasive capacity of autophagy-deficient cells, indicating these cells fail to secrete factors required for RAS-driven invasion. Reduced autophagy diminishes the secretion of the pro-migratory cytokine IL6, which is necessary to restore invasion of autophagy-deficient cells. Moreover, autophagy-deficient cells exhibit reduced levels of MMP2 and WNT5A. These results support a previously unrecognized function for autophagy in promoting cancer cell invasion via the coordinate production of multiple secreted factors

Autophagy-dependent production of secreted factors facilitates oncogenic RAS-driven invasion.

UnlabelledThe tumor-promoting functions of autophagy are primarily attributed to its ability to promote cancer cell survival. However, emerging evidence suggests that autophagy plays other roles during tumorigenesis. Here, we uncover that autophagy promotes oncogenic RAS-driven invasion. In epithelial cells transformed with oncogenic RAS, depletion of autophagy-related genes suppresses invasion in three-dimensional culture, decreases cell motility, and reduces pulmonary metastases in vivo. Treatment with conditioned media from autophagy-competent cells rescues the invasive capacity of autophagy-deficient cells, indicating that these cells fail to secrete factors required for RAS-driven invasion. Reduced autophagy diminishes the secretion of the promigratory cytokine interleukin-6 (IL-6), which is necessary to restore invasion of autophagy-deficient cells. Moreover, autophagy-deficient cells exhibit reduced levels of matrix metalloproteinase 2 and WNT5A. These results support a previously unrecognized function for autophagy in promoting cancer cell invasion via the coordinate production of multiple secreted factors.SignificanceOur results delineate a previously unrecognized function for autophagy in facilitating oncogenic RAS-driven invasion. We demonstrate that an intact autophagy pathway is required for the elaboration of multiple secreted factors favoring invasion, including IL-6

Autophagic Degradation of NBR1 Restricts Metastatic Outgrowth during Mammary Tumor Progression

Although autophagy is being pursued as a therapeutic target in clinical oncology trials, its effects on metastasis, the principal cause of cancer mortality, remain unclear. Here, we utilize mammary cancer models to temporally delete essential autophagy regulators during carcinoma progression. Though genetic ablation of autophagy strongly attenuates primary mammary tumor growth, impaired autophagy promotes spontaneous metastasis and enables the outgrowth of disseminated tumor cells into overt macro-metastases. Transcriptomic analysis reveals that autophagy deficiency elicits a subpopulation of otherwise luminal tumor cells exhibiting basal differentiation traits, which is reversed upon preventing accumulation of the autophagy cargo receptor, Neighbor to BRCA1 (NBR1). Furthermore, pharmacological and genetic induction of autophagy suppresses pro-metastatic differentiation and metastatic outgrowth. Analysis of human breast cancer data reveal that autophagy gene expression inversely correlates with pro-metastatic differentiation signatures and predicts overall and distant metastasis-free survival. Overall, these findings highlight autophagy-dependent control of NBR1 as a key determinant of metastatic progression

Extracellular vesicle and particle isolation from human and murine cell lines, tissues, and bodily fluids

We developed a modified protocol, based on differential ultracentrifugation (dUC), to isolate extracellular vesicles and particles (specifically exomeres) (EVPs) from various human and murine sources, including cell lines, surgically resected tumors and adjacent tissues, and bodily fluids, such as blood, lymphatic fluid, and bile. The diversity of these samples requires robust and highly reproducible protocols and refined isolation technology, such as asymmetric-flow field-flow fractionation (AF4). Our isolation protocol allows for preparation of EVPs for various downstream applications, including proteomic profiling. For complete details on the use and execution of this protocol, please refer to Hoshino et al. (2020).Funding agencies: he National Cancer Institute, United States CA224175 (D.L.), CA210240 (D.L.), CA232093(D.L.), CA163117 and CA207983 (D.L.), CA163120 (D.L.), CA169416 (D.L.), CA169538 (D.L.),CA218513 (D.L., H.Z.) and AI144301 (D.L., V.P.); the United States Department of Defense, UnitedStates W81XWH-13-1-0425 (D.L.), W81XWH-13-1-0427, W81XWH-13-1-0249 (D.L.), and W81XWH-14-1-0199 (D.L.); National Institutes of Health, United States/WCM CTSC, United States (NIH/NCATS (UL1TR00457) (H.Z.); NIH/NCATS (UL1TR002384) (D.L. and H.Z.); the Hartwell Foundation,United States (D.L.); the Thompson Family Foundation (D.L., D.K.); the STARR Consortium I9-A9-056(D.L., H.Z.) and I8-A8-123 (D.L.); the Pediatric Oncology Experimental Therapeutics Investigator’sConsortium (D.L., T.M.T.); Alex’s Lemonade Stand Foundation, United Sates (D.L.); the Breast Can-cer Research Foundation, United States (D.L.); the Feldstein Medical Foundation (D.L.); the TortolaniFoundation (D.L.); the Clinical &amp; Translational Science Center (D.L., H.Z.); the Mary Kay Ash Chari-table Foundation (D.L., I.M.); the Malcolm Hewitt Weiner Foundation (D.L.); the Manning Founda-tion (DL., A.H.); the Daniel P. and Nancy C. Paduano Family Foundation (D.L); the James PaduanoFoundation (D.L.); the Sohn Foundation (D.L.); the AHEPA Vth District Cancer Research Foundation(D.L., L.B., S.L.); the Daedalus Fund Selma and Lawrence Ruben Science to Industry Bridge Award(D.L.); the Children’s Cancer and Blood Foundation, United States (D.L.); Susan G. Komen Postdoc-toral Fellowship PDF15331556, JST PRESTO, Japan 30021, and JSPS KAKENHI, Japan JP19K23743(A.H.); the National Research Foundation of Korea, South Korea (NRF) grant funded by the Korea government (MSIT) (2019R1C1C1006709, 2018R1A5A2025079, and 2020M3F7A1094093); a grantof the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Insti-tute (KHIDI), funded by the Ministry of Health &amp; Welfare, South Korea (KHIDIHI19C1015010020);"The Alchemist Project" through the Ministry of Trade, Industry and Energy (MOTIE, Korea)(20012443), and Severance Hospital Research fund for Clinical excellence (SHRC) (C-2020-0032and C-2020-0025) (H.S.K.); The Swedish Cancer Society Pancreatic Cancer Fellowship, the Lions In-ternational Postdoctoral fellowship and the Sweden-America stipend (L.B.); the Sa ̃ o Paulo ResearchFoundation (FAPESP) Sa ̃ o Paulo, Brazil (2017/07117-7) (G.C.T); the Swiss National Science Founda-tion (SFNS), Switzerland Postdoc Mobility grant (P2SKP3_174785 and P400PB_186791) (F.A.V.); andthe DoD PRCRP Horizon Award, United States (W81XWH-19-PRCRP-HA) (S.L).</p