Epithelial-mesenchymal transition (EMT) is a transient, reversible process of cell de-differentiation where cancer cells transit between various stages of an EMT continuum, including epithelial, ...partial EMT, and mesenchymal cell states. We have employed Tamoxifen-inducible dual recombinase lineage tracing systems combined with live imaging and 5-cell RNA sequencing to track cancer cells undergoing partial or full EMT in the MMTV-PyMT mouse model of metastatic breast cancer. In primary tumors, cancer cells infrequently undergo EMT and mostly transition between epithelial and partial EMT states but rarely reach full EMT. Cells undergoing partial EMT contribute to lung metastasis and chemoresistance, whereas full EMT cells mostly retain a mesenchymal phenotype and fail to colonize the lungs. However, full EMT cancer cells are enriched in recurrent tumors upon chemotherapy. Hence, cancer cells in various stages of the EMT continuum differentially contribute to hallmarks of breast cancer malignancy, such as tumor invasion, metastasis, and chemoresistance.
Display omitted
•Lineage tracing of partial and full EMT cells in breast cancer metastasis•Partial EMT cells cycle between hybrid EMT and epithelial stages•Partial, but not full, EMT cells are required for metastasis formation•Both partial and full EMT cells contribute to chemoresistance
Lüönd et al. establish genetic lineage tracing systems to monitor mammary tumor cells undergoing early partial and late full epithelial-mesenchymal transition (EMT). They demonstrate that partial EMT cells, but not full EMT cells, are required for lung metastasis, while both contribute to the development of chemoresistance.
Understanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In this study, a combination of shRNA‐mediated synthetic ...lethality screening and transcriptomic analysis revealed the transcription factors YAP/TAZ as key drivers of Sorafenib resistance in hepatocellular carcinoma (HCC) by repressing Sorafenib‐induced ferroptosis. Mechanistically, in a TEAD‐dependent manner, YAP/TAZ induce the expression of SLC7A11, a key transporter maintaining intracellular glutathione homeostasis, thus enabling HCC cells to overcome Sorafenib‐induced ferroptosis. At the same time, YAP/TAZ sustain the protein stability, nuclear localization, and transcriptional activity of ATF4 which in turn cooperates to induce SLC7A11 expression. Our study uncovers a critical role of YAP/TAZ in the repression of ferroptosis and thus in the establishment of Sorafenib resistance in HCC, highlighting YAP/TAZ‐based rewiring strategies as potential approaches to overcome HCC therapy resistance.
SYNOPSIS
Resistance to therapy occurs in most liver cancer patients treated with Sorafenib, and patients succumb to the disease. A synthetic lethal screen identified a regulatory circuit, which prevents ferroptosis and promotes cancer cell survival, thus promoting resistance to Sorafenib.
The transcription factors YAP and TAZ stabilize ATF4 by promoting its nuclear import to cooperatively induce expression of SLC7A11, a cystine importer critical for glutathione synthesis.
Glutathione synthesis and homeostasis are required to repress ferroptosis and to maintain Sorafenib resistance in liver cancer cells.
Inhibition of Glutathione synthesis re‐sensitizes Sorafenib‐resistant cancer cells to Sorafenib therapy, which then induces ferroptosis and represses tumor growth in murine liver cancer models.
Pharmacological repression of the anti‐oxidant pathways regulated by YAP/TAZ and ATF4 could re‐sensitize therapy‐resistant liver cancers to Sorafenib treatment.
Resistance to therapy occurs in most liver cancer patients treated with Sorafenib, and patients succumb to the disease. A synthetic lethal screen identified a regulatory circuit, which prevents ferroptosis and promotes cancer cell survival, thus promoting resistance to Sorafenib.
Abstract
Understanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In hepatocellular carcinoma (HCC), aberrant ...expression of hypoxia-inducible factor 1 α (HIF1α) and increased aerobic glycolysis metabolism are drivers of resistance to therapy with the multi-kinase inhibitor Sorafenib. However, it has remained unknown how HIF1α is activated and how its activity and the subsequent induction of aerobic glycolysis promote Sorafenib resistance in HCC. Here, we report the ubiquitin-specific peptidase USP29 as a new regulator of HIF1α and of aerobic glycolysis during the development of Sorafenib resistance in HCC. In particular, we identified USP29 as a critical deubiquitylase (DUB) of HIF1α, which directly deubiquitylates and stabilizes HIF1α and, thus, promotes its transcriptional activity. Among the transcriptional targets of HIF1α is the gene encoding hexokinase 2 (HK2), a key enzyme of the glycolytic pathway. The absence of USP29, and thus of HIF1α transcriptional activity, reduces the levels of aerobic glycolysis and restores sensitivity to Sorafenib in Sorafenib-resistant HCC cells in vitro and in xenograft transplantation mouse models in vivo. Notably, the absence of USP29 and high HK2 expression levels correlate with the response of HCC patients to Sorafenib therapy. Together, the data demonstrate that, as a DUB of HIF1α, USP29 promotes Sorafenib resistance in HCC cells, in parts by upregulating glycolysis, thereby opening new avenues for therapeutically targeting Sorafenib-resistant HCC in patients.
Phosphorylation of the transcriptional coactivator YAP1 is a key event in defining Hippo signaling outputs. Previous studies demonstrated that phosphorylation of YAP1 at serine 127 (S127) sequesters ...YAP1 in the cytoplasm and consequently inhibits YAP1 transcriptional activity. Mammalian tissue-culture experiments suggest that downstream of MST1/2 signaling, LATS1/2 function as YAP1-S127 kinases. However, studies of Mst1/2 knockout mouse models revealed that the identity of the physiological YAP1-S127 kinase(s) in certain tissues, such as the intestine, remains unknown.
We show that mammalian NDR1/2 kinases phosphorylate YAP1 on S127 and thereby negatively regulate YAP1 activity in tissue-cultured cells. By studying NDR1/2-deficient mice, we demonstrate the in vivo relevance of NDR1/2-mediated regulation of YAP1. Specifically, upon loss of NDR1/2 in the intestinal epithelium, endogenous S127 phosphorylation is decreased whereas total YAP1 levels are increased. Significantly, ablation of NDR1/2 from the intestinal epithelium renders mice exquisitely sensitive to chemically induced colon carcinogenesis. Analysis of human colon cancer samples further revealed that NDR2 and YAP1 protein expression are inversely correlated in the majority of samples with high YAP1 expression. Collectively, we report NDR1/2 as physiological YAP1-S127 kinases that might function as tumor suppressors upstream of YAP1 in human colorectal cancer.
We establish mammalian NDR1/2 as bona fide kinases that target YAP1 on S127 in vitro and in vivo. Our findings therefore have important implications for a broad range of research efforts aimed at decoding and eventually manipulating YAP1 biology in cancer settings, regenerative medicine, and possibly also noncancer human diseases.
Display omitted
•Mammalian NDR kinases phosphorylate YAP1 on serine 127•Phosphorylation of YAP1 by NDR kinases regulates YAP1 activity in vivo•NDR kinases function as tumor suppressors in the intestinal epithelium•Ndr knockout mice represent the first animal model of a direct S127 kinase
Phosphorylation of the Hippo pathway effector YAP1 contributes to tissue homeostasis. However, the identity of the YAP1 kinase in the intestine remains unknown. Here, Zhang et al. report NDR as a physiological YAP1 kinase, restricting YAP1’s activity in the intestine and hence establishing the first mouse model of a direct YAP1-S127 kinase.
Ferroptosis is an emerging form of regulated cell death in an oxidative stress- and iron-dependent manner, primarily induced by the over-production of reactive oxygen species (ROS). Manipulation of ...ferroptosis has been considered a promising therapeutic approach to inhibit liver tumor growth. Nevertheless, the development of resistance to ferroptosis in liver cancer poses a significant challenge in cancer treatment. Post-translational modifications (PTMs) are crucial enzymatic catalytic reactions that covalently regulate protein conformation, stability and cellular activities. Additionally, PTMs play pivotal roles in various biological processes and divergent programmed cell death, including ferroptosis. Importantly, key PTMs regulators involved in ferroptosis have been identified as potential targets for cancer therapy. PTMs function of two proteins, SLC7A11, GPX4 involved in ferroptosis resistance have been extensively investigated in recent years. This review will summarize the roles of PTMs in ferroptosis-related proteins in hepatocellular carcinoma (HCC) treatment.
Approaches to study therapy resistance in HCC (hepatocellular carcinoma) are limited, especially when using HCC models in vitro. Here, we present a protocol to establish an in vitro ...Sorafenib-resistant human HCC cell model and conduct an shRNA-mediated synthetic lethal screen in established Sorafenib-resistant HCC cell lines to identify critical regulators of Sorafenib resistance. We describe steps for RNA sequencing and functional analysis to reveal the mode of action of potential candidates in conferring therapy resistance to HCC cells.
For complete details on the use and execution of this protocol, please refer to Gao et al. (2021a)1 and Gao et al. (2021b).2
Display omitted
•Establishment of in vitro Sorafenib-resistant HCC cell models•Establishment of intrinsic or acquired drug-resistant cancer cell lines•Global transcriptomic analysis by RNA sequencing•Synthetic lethal screen using genome-wide pooled lentiviral shRNA libraries
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Approaches to study therapy resistance in HCC (hepatocellular carcinoma) are limited, especially when using HCC models in vitro. Here, we present a protocol to establish an in vitro Sorafenib-resistant human HCC cell model and conduct an shRNA-mediated synthetic lethal screen in established Sorafenib-resistant HCC cell lines to identify critical regulators of Sorafenib resistance. We describe steps for RNA sequencing and functional analysis to reveal the mode of action of potential candidates in conferring therapy resistance to HCC cells.
Autophagy perturbation represents an emerging therapeutic strategy in cancer. Although LATS1 and LATS2 kinases, core components of the mammalian Hippo pathway, have been shown to exert tumor ...suppressive activities, here we report a pro-survival role of LATS1 but not LATS2 in hepatocellular carcinoma (HCC) cells. Specifically, LATS1 restricts lethal autophagy in HCC cells induced by sorafenib, the standard of care for advanced HCC patients. Notably, autophagy regulation by LATS1 is independent of its kinase activity. Instead, LATS1 stabilizes the autophagy core-machinery component Beclin-1 by promoting K27-linked ubiquitination at lysine residues K32 and K263 on Beclin-1. Consequently, ubiquitination of Beclin-1 negatively regulates autophagy by promoting inactive dimer formation of Beclin-1. Our study highlights a functional diversity between LATS1 and LATS2, and uncovers a scaffolding role of LATS1 in mediating a cross-talk between the Hippo signaling pathway and autophagy.
Understanding the mechanisms of evasive resistance in cancer is of great importance to develop efficient therapies. Analyzing the molecular mechanisms underlying therapy resistance of hepatocellular ...carcinoma (HCC), we have discovered a kinase-activity independent role of LATS1 (large tumor suppressor) but not LATS2 in regulating sorafenib-induced lethal autophagy in HCC. We have found that the autophagy regulatory role of LATS1 is a general phenomenon in response to various stimuli of autophagy induction which relies on a LATS1-specific protein domain. Mechanistically, the autophagy regulatory role of LATS1 is coupled with Beclin-1 (BECN1) K27-linked ubiquitination and BECN1 self-dimerization. Our study highlights a LATS1-mediated non-classical interaction between the Hippo signaling pathway and autophagy in therapy response and carcinogenesis.
Organ development is precisely guided by spatiotemporal cross-talks between a variety of signaling pathways regulating cell differentiation, proliferation, growth arrest and physiological cell death. ...Aberrant signaling inputs invariably lead to tissue dysfunction and to certain conditions, even malignant transformation. In this review, we focus on the functional interplay between the Hippo signaling pathway and autophagy in normal tissue homeostasis and in malignant tumor progression. Mounting experimental evidence for the regulation of cancer cell malignancy and therapy resistance by the functional cross-talk between Hippo signaling and autophagy highlights this signaling axis as a suitable therapeutic target to combat cancer.
New therapeutic targets are needed that circumvent inherent therapeutic resistance of glioblastoma multiforme (GBM). Here, we report such a candidate target in the uncharacterized adaptor protein ...hMOB3, which we show is upregulated in GBM. In a search for its biochemical function, we found that hMOB3 specifically interacts with MST1 kinase in response to apoptotic stimuli and cell-cell contact. Moreover, hMOB3 negatively regulated apoptotic signaling by MST1 in GBM cells by inhibiting the MST1 cleavage-based activation process. Physical interaction between hMOB3 and MST1 was essential for this process. In vivo investigations established that hMOB3 sustains GBM cell growth at high cell density and promotes tumorigenesis. Our results suggest hMOB3 as a candidate therapeutic target for the treatment of malignant gliomas.
PDF
