Syn Kok Yeo

Autophagy Differentially Regulates Distinct Breast Cancer Stem-like Cells in Murine Models via EGFR/Stat3 and Tgfβ/Smad Signaling

Cancer stem-like cells contribute to tumor heterogeneity and have been implicated in disease relapse and drug resistance. Here we show the coexistence of distinct breast cancer stem-like cells (BCSC) as identified by ALDH(+) and CD29(hi)CD61(+) markers, respectively, in murine models of breast cancer. While both BCSC exhibit enhanced tumor-initiating potential, CD29(hi)CD61(+) BCSC displayed increased invasive abilities and higher expression of epithelial-to-mesenchymal transition and mammary stem cell-associated genes, whereas ALDH(+) BCSC were more closely associated with luminal progenitors. Attenuating the autophagy regulator FIP200 diminished the tumor-initiating properties of both ALDH(+) and CD29(hi)CD61(+) BCSC, as achieved by impairing either the Stat3 or TGFβ/Smad pathways, respectively. Furthermore, combining the Stat3 inhibitor Stattic and the Tgfβ-R1 inhibitor LY-2157299 inhibited the formation of both epithelial and mesenchymal BCSC colonies. In vivo, this combination treatment was sufficient to limit tumor growth and reduce BCSC number. Overall, our findings reveal a differential dependence of heterogeneous BCSC populations on divergent signaling pathways, with implications on how to tailor drug combinations to improve therapeutic efficacy. Cancer Res; 76(11); 3397-410. ©2016 AACR.

Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model

Autophagy is an evolutionarily conserved cellular process controlled through a set of essential autophagy genes (Atgs). However, there is increasing evidence that most, if not all, Atgs also possess functions independent of their requirement in canonical autophagy, making it difficult to distinguish the contributions of autophagy-dependent or -independent functions of a particular Atg to various biological processes. To distinguish these functions for FIP200 (FAK family-interacting protein of 200 kDa), an Atg in autophagy induction, we examined FIP200 interaction with its autophagy partner, Atg13. We found that residues 582-585 (LQFL) in FIP200 are required for interaction with Atg13, and mutation of these residues to AAAA (designated the FIP200-4A mutant) abolished its canonical autophagy function in vitro. Furthermore, we created a FIP200-4A mutant knock-in mouse model and found that specifically blocking FIP200 interaction with Atg13 abolishes autophagy in vivo, providing direct support for the essential role of the ULK1/Atg13/FIP200/Atg101 complex in the process beyond previous studies relying on the complete knockout of individual components. Analysis of the new mouse model showed that nonautophagic functions of FIP200 are sufficient to fully support embryogenesis by maintaining a protective role in TNFα-induced apoptosis. However, FIP200-mediated canonical autophagy is required to support neonatal survival and tumor cell growth. These studies provide the first genetic evidence linking an Atgs autophagy and nonautophagic functions to different biological processes in vivo.

Elevated p62/SQSTM1 determines the fate of autophagy-deficient neural stem cells by increasing superoxide

Comparison of autophagy inhibition by deletion of Atg5, Atg16L1, Atg7, or Fip200 reveals a critical role for increased p62 in determining the fate of autophagy-deficient neural stem cells by controlling intracellular superoxide.

Autophagy inhibition re-sensitizes pulse stimulation-selected paclitaxel-resistant triple negative breast cancer cells to chemotherapy-induced apoptosis.

Chemotherapy is the mainstay of systemic treatment for triple negative breast cancer (TNBC); however, the development of drug resistance limits its effectiveness. Therefore, we investigated the underlying mechanism for drug resistance and potential approaches to overcome it for a more effective treatment for TNBCs. Using a pulse-stimulated selection strategy to mimic chemotherapy administration in the clinic, we developed a new paclitaxel-resistant MDA-MB-231 cell line and analyzed these cells for changes in autophagy activity, and the role and mechanisms of the increased autophagy in promoting drug resistance were determined. We found that the pulse-stimulated selection strategy with paclitaxel resulted in MDA-MB-231 variant cells with enhanced resistance to paclitaxel. These resistant cells were found to have enhanced basal autophagy activity, which confers a cytoprotective function under paclitaxel treatment stress. Inhibition of autophagy enhanced paclitaxel-induced cell death in these paclitaxel-resistant cells. We further revealed that up-regulated autophagy in resistant cells enhanced the clearance of damaged mitochondria. Last, we showed that the paclitaxel-resistant cancer cells acquired cross resistance to epirubicin and cisplatin. Together, these results suggest that combining autophagy inhibition with chemotherapy may be an effective strategy to improve treatment outcome in paclitaxel-resistant TNBC patients.

Autophagy gene FIP200 in neural progenitors non–cell autonomously controls differentiation by regulating microglia

Recent studies have shown important roles for autophagy genes in the regulation of different tissue stem cells, including neural stem/progenitor cells (NSCs). However, little is known about whether autophagy can regulate NSCs through cell-extrinsic mechanisms. Here, we show that deletion of an essential autophagy gene, FIP200, in NSCs increased expression of Ccl5 and Cxcl10 in a p53-independent manner, mediating increased infiltration of microglia into the subventricular zone of both FIP200hGFAP conditional knockout (cKO) and FIP200;p53hGFAP 2cKO mice. The microglia exhibited an activated M1 phenotype consistent with their potential to inhibit differentiation of FIP200-null NSCs. Blocking either microglia infiltration or activation rescued the deficient differentiation of FIP200-null NSCs from FIP200;p53hGFAP 2cKO mice. Lastly, we showed that increased chemokine expression in FIP200-null NSCs was induced by abnormal p62 aggregate formation and activation of NF-&kgr;B signaling. Our results suggest that autophagy plays a crucial role in regulating neurogenesis and restricting local immune response in postnatal NSCs through non–cell autonomous mechanisms.

Breast Cancer: Multiple Subtypes within a Tumor?

Breast cancer is a heterogeneous disease, and stratification of tumors is paramount to achieve better clinical outcomes. While it is common to stratify and treat breast tumors as a single entity, insights from studies on intratumoral heterogeneity and cancer stem cells raise the possibility that multiple breast cancer subtypes may coexist within a tumor. A role for plasticity in driving dynamic conversions between breast cancer subtypes is proposed, and the clinical implications include a need for combinatorial therapeutic strategies that account for the discrete disease entities and their plasticity. Accordingly, the advent of single-cell technologies will be crucial in enabling the diagnosis and stratification of distinct disease subtypes down to the cellular level.

Hierarchical heterogeneity in mammary tumors and its regulation by autophagy.

ABSTRACT Intra-tumor heterogeneity can be attributed in part to the ability of tumor cells to acquire traits associated with less differentiated cells. In MMTV-PyMT mammary tumors, this hierarchical heterogeneity can be illustrated with the use of ITGB1/CD29hi ITGB3/CD61+ markers to enrich for mammary stem-like cells and ALDH+ to identify luminal progenitor-like cells. Macroautophagy/autophagy appears to be important for maintaining the cancer stem-like traits of both these populations. Interestingly, the regulation of these distinct cancer stem-like cells by autophagy occurs through EGFR-STAT3 and TGFB/TGF-β-SMAD pathways, respectively. These findings indicate that autophagy plays a significant role in cancer stem-like cells, and distinct cancer stem-like cells within a tumor may require different treatment modalities.

Abstract B21: Improved efficacy of mitochondrial disrupting agents upon inhibition of autophagy in a mouse model of BRCA1-deficient breast cancer

Breast cancer is a heterogeneous disease, and stratification of patients is fundamental to the success of treatment modalities. Breast tumors deficient in BRCA1 are mostly associated with basal-like breast cancers, and targeted therapeutics for this disease subtype are still lacking. In order to address whether autophagy inhibition will be effective in BRCA1-deficient mammary tumors, we generated mice with conditional deletion of an essential autophagy gene FIP200 along with Brca1 and Trp53, through utilization of the K14-Cre transgene. We found that FIP200 deletion suppressed tumorigenesis in the BRCA1-deficient model when compared to wild-type and heterozygous FIP200 controls. However, in contrast to previous studies in the MMTV-PyMT model, tumor growth and the distribution of histologic subtypes were not affected by loss of FIP200. Interestingly, loss of FIP200 decreased mitochondrial mass and oxidative respiratory capacity of these tumor cells, along with a decrease in the phosphorylation of mTOR substrates and transcript levels of genes involved in mitochondrial biogenesis. Importantly, we observed an increased sensitivity to mitochondrial disrupting agents upon loss of FIP200. Consequently, our data showed that combination of an autophagy inhibitor, Spautin-1, along with a mitochondrial complex I inhibitor, metformin, was more effective in limiting oxidative respiratory capacity, colony-forming ability, and tumor growth. Altogether, our results indicate that inhibition of autophagy can increase the benefits of metformin treatment in BRCA1-deficient breast cancers. Citation Format: Syn Kok Yeo, Ritama Paul, Michael Haas, Chenran Wang, Jun-Lin Guan. Improved efficacy of mitochondrial disrupting agents upon inhibition of autophagy in a mouse model of BRCA1-deficient breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B21.

Improved efficacy of mitochondrial disrupting agents upon inhibition of autophagy in a mouse model of BRCA1-deficient breast cancer

ABSTRACT Breast cancer is a heterogeneous disease, and stratification of patients is fundamental to the success of treatment modalities. Breast tumors deficient in BRCA1 are mostly associated with basal-like breast cancers and targeted therapeutics for this disease subtype are still lacking. In order to address whether macroautophagy/autophagy inhibition will be effective in BRCA1-deficient mammary tumors, we generated mice with conditional deletion of an essential autophagy gene, Rb1cc1, along with Brca1 and Trp53, through utilization of the K14-Cre transgene. We found that Rb1cc1 deletion suppressed tumorigenesis in the BRCA1-deficient model when compared to wild type and heterozygous Rb1cc1 controls. However, in contrast to previous studies in the mouse mammary tumor virus (MMTV)-polyoma middle T antigen (PyMT) model, tumor growth and the distribution of histological subtypes were not affected by loss of RB1CC1. Interestingly, loss of RB1CC1 decreased mitochondrial mass and oxidative respiratory capacity of these tumor cells, along with a decrease in the phosphorylation of MTOR substrates and transcript levels of genes involved in mitochondrial biogenesis. Importantly, we observed an increased sensitivity to mitochondrial disrupting agents upon loss of RB1CC1. Consequently, our data showed that combination of an autophagy inhibitor, spautin-1, along with a mitochondrial complex I inhibitor, metformin, was more effective in limiting oxidative respiratory capacity, colony-forming ability and tumor growth. Altogether, our results indicate that inhibition of autophagy can increase the benefits of metformin treatment in BRCA1-deficient breast cancers.