Protocol to screen for Sorafenib resistance regulators using pooled lentiviral shRNA library and a Sorafenib-resistant hepatocellular carcinoma cell model.

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.

Tracking and characterization of partial and full epithelial-mesenchymal transition cells in a mouse model of metastatic breast cancer.

The various stages of epithelial-mesenchymal transition (EMT) generate phenotypically heterogeneous populations of cells. Here, we detail a dual recombinase lineage tracing system using a transgenic mouse model of metastatic breast cancer to trace and characterize breast cancer cells at different EMT stages. We describe analytical steps to label cancer cells at an early partial or a late full EMT state, followed by tracking their behavior in tumor slice cultures. We then characterize their transcriptome by five-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Luond et al. (2021).

Hierarchy of TGFß/SMAD, Hippo/YAP/TAZ, and Wnt/ß-catenin signaling in melanoma phenotype switching.

In melanoma, a switch from a proliferative melanocytic to an invasive mesenchymal phenotype is based on dramatic transcriptional reprogramming which involves complex interactions between a variety of signaling pathways and their downstream transcriptional regulators. TGFß/SMAD, Hippo/YAP/TAZ, and Wnt/ß-catenin signaling pathways are major inducers of transcriptional reprogramming and converge at several levels. Here, we report that TGFß/SMAD, YAP/TAZ, and ß-catenin are all required for a proliferative-to-invasive phenotype switch. Loss and gain of function experimentation, global gene expression analysis, and computational nested effects models revealed the hierarchy between these signaling pathways and identified shared target genes. SMAD-mediated transcription at the top of the hierarchy leads to the activation of YAP/TAZ and of ß-catenin, with YAP/TAZ governing an essential subprogram of TGFß-induced phenotype switching. Wnt/ß-catenin signaling is situated further downstream and exerts a dual role: it promotes the proliferative, differentiated melanoma cell phenotype and it is essential but not sufficient for SMAD or YAP/TAZ-induced phenotype switching. The results identify epistatic interactions among the signaling pathways underlying melanoma phenotype switching and highlight the priorities in targets for melanoma therapy.

Distinct contributions of partial and full EMT to breast cancer malignancy.

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.

The long non-coding RNA ET-20 mediates EMT by impairing desmosomes in breast cancer cells.

The vast majority of breast cancer-associated deaths are due to metastatic spread of cancer cells, a process aided by epithelial-to-mesenchymal transition (EMT). Mounting evidence has indicated that long non-coding RNAs (lncRNAs) also contribute to tumor progression. We report the identification of 114 novel lncRNAs that change their expression during TGFß-induced EMT in murine breast cancer cells (referred to as EMT-associated transcripts; ETs). Of these, the ET-20 gene localizes in antisense orientation within the tenascin C (Tnc) gene locus. TNC is an extracellular matrix protein that is critical for EMT and metastasis formation. Both ET-20 and Tnc are regulated by the EMT master transcription factor Sox4. Notably, ablation of ET-20 lncRNA effectively blocks Tnc expression and with it EMT. Mechanistically, ET-20 interacts with desmosomal proteins, thereby impairing epithelial desmosomes and promoting EMT. A short transcript variant of ET-20 is shown to be upregulated in invasive human breast cancer cell lines, where it also promotes EMT. Targeting ET-20 appears to be a therapeutically attractive lead to restrain EMT and breast cancer metastasis in addition to its potential utility as a biomarker for invasive breast cancer.

YAP/TAZ and ATF4 drive resistance to Sorafenib in hepatocellular carcinoma by preventing ferroptosis.

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.

The interactions of Bcl9/Bcl9L with ß-catenin and Pygopus promote breast cancer growth, invasion, and metastasis.

Canonical Wnt/ß-catenin signaling is an established regulator of cellular state and its critical contributions to tumor initiation, malignant tumor progression and metastasis formation have been demonstrated in various cancer types. Here, we investigated how the binding of ß-catenin to the transcriptional coactivators B-cell CLL/lymphoma 9 (Bcl9) and Bcl9-Like (Bcl9L) affected mammary gland carcinogenesis in the MMTV-PyMT transgenic mouse model of metastatic breast cancer. Conditional knockout of both Bcl9 and Bcl9L resulted into tumor cell death. In contrast, disrupting the interaction of Bcl9/Bcl9L with ß-catenin, either by deletion of their HD2 domains or by a point mutation in the N-terminal domain of ß-catenin (D164A), diminished primary tumor growth and tumor cell proliferation and reduced tumor cell invasion and lung metastasis. In comparison, the disruption of HD1 domain-mediated binding of Bcl9/Bcl9L to Pygopus had only moderate effects. Interestingly, interfering with the ß-catenin-Bcl9/Bcl9L-Pygo chain of adapters only partially impaired the transcriptional response of mammary tumor cells to Wnt3a and TGFß treatments. Together, the results indicate that Bcl9/Bcl9L modulate but are not critically required for canonical Wnt signaling in its contribution to breast cancer growth and malignant progression, a notion consistent with the "just-right" hypothesis of Wnt-driven tumor progression.

Parsing ß-catenin's cell adhesion and Wnt signaling functions in malignant mammary tumor progression.

During malignant progression, epithelial cancer cells dissolve their cell-cell adhesion and gain invasive features. By virtue of its dual function, ß-catenin contributes to cadherin-mediated cell-cell adhesion, and it determines the transcriptional output of Wnt signaling: via its N terminus, it recruits the signaling coactivators Bcl9 and Pygopus, and via the C terminus, it interacts with the general transcriptional machinery. This duality confounds the simple loss-of-function analysis of Wnt signaling in cancer progression. In many cancer types including breast cancer, the functional contribution of ß-catenin's transcriptional activities, as compared to its adhesion functions, to tumor progression has remained elusive. Employing the mouse mammary tumor virus (MMTV)-PyMT mouse model of metastatic breast cancer, we compared the complete elimination of ß-catenin with the specific ablation of its signaling outputs in mammary tumor cells. Notably, the complete lack of ß-catenin resulted in massive apoptosis of mammary tumor cells. In contrast, the loss of ß-catenin's transcriptional activity resulted in a reduction of primary tumor growth, tumor invasion, and metastasis formation in vivo. These phenotypic changes were reflected by stalled cell cycle progression and diminished epithelial-mesenchymal transition (EMT) and cell migration of breast cancer cells in vitro. Transcriptome analysis revealed subsets of genes which were specifically regulated by ß-catenin's transcriptional activities upon stimulation with Wnt3a or during TGF-ß-induced EMT. Our results uncouple the signaling from the adhesion function of ß-catenin and underline the importance of Wnt/ß-catenin-dependent transcription in malignant tumor progression of breast cancer.

USP29-mediated HIF1α stabilization is associated with Sorafenib resistance of hepatocellular carcinoma cells by upregulating glycolysis.

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.

Transcriptional Enhancer Factor Domain Family member 4 Exerts an Oncogenic Role in Hepatocellular Carcinoma by Hippo-Independent Regulation of Heat Shock Protein 70 Family Members.

Transcriptional enhancer factor domain family member 4 (TEAD4) is a downstream effector of the conserved Hippo signaling pathway, regulating the expression of genes involved in cell proliferation and differentiation. It is up-regulated in several cancer types and is associated with metastasis and poor prognosis. However, its role in hepatocellular carcinoma (HCC) remains largely unexplored. Using data from The Cancer Genome Atlas, we found that TEAD4 was overexpressed in HCC and was associated with aggressive HCC features and worse outcome. Overexpression of TEAD4 significantly increased proliferation and migration rates in HCC cells in vitro as well as tumor growth in vivo. Additionally, RNA sequencing analysis of TEAD4-overexpressing HCC cells demonstrated that TEAD4 overexpression was associated with the up-regulation of genes involved in epithelial-to-mesenchymal transition, proliferation, and protein-folding pathways. Among the most up-regulated genes following TEAD4 overexpression were the 70-kDa heat shock protein (HSP70) family members HSPA6 and HSPA1A. Chromatin immunoprecipitation-quantitative real-time polymerase chain reaction experiments demonstrated that TEAD4 regulates HSPA6 and HSPA1A expression by directly binding to their promoter and enhancer regions. The pharmacologic inhibition of HSP70 expression in TEAD4-overexpressing cells reduced the effect of TEAD4 on cell proliferation. Finally, by overexpressing TEAD4 in yes-associated protein (YAP)/transcriptional coactivator with PDZ binding motif (TAZ)-knockdown HCC cells, we showed that the effect of TEAD4 on cell proliferation and its regulation of HSP70 expression does not require YAP and TAZ, the main effectors of the Hippo signaling pathway. Conclusion: A novel Hippo-independent mechanism for TEAD4 promotes cell proliferation and tumor growth in HCC by directly regulating HSP70 family members.

Breast cancer as an example of tumour heterogeneity and tumour cell plasticity during malignant progression.

Heterogeneity within a tumour increases its ability to adapt to constantly changing constraints, but adversely affects a patient's prognosis, therapy response and clinical outcome. Intratumoural heterogeneity results from a combination of extrinsic factors from the tumour microenvironment and intrinsic parameters from the cancer cells themselves, including their genetic, epigenetic and transcriptomic traits, their ability to proliferate, migrate and invade, and their stemness and plasticity attributes. Cell plasticity constitutes the ability of cancer cells to rapidly reprogramme their gene expression repertoire, to change their behaviour and identities, and to adapt to microenvironmental cues. These features also directly contribute to tumour heterogeneity and are critical for malignant tumour progression. In this article, we use breast cancer as an example of the origins of tumour heterogeneity (in particular, the mutational spectrum and clonal evolution of progressing tumours) and of tumour cell plasticity (in particular, that shown by tumour cells undergoing epithelial-to-mesenchymal transition), as well as considering interclonal cooperativity and cell plasticity as sources of cancer cell heterogeneity. We review current knowledge on the functional contribution of cell plasticity and tumour heterogeneity to malignant tumour progression, metastasis formation and therapy resistance.

OBF1 and Oct factors control the germinal center transcriptional program.

OBF1 is a specific coactivator of the POU family transcription factors OCT1 and OCT2. OBF1 and OCT2 are B cell-specific and indispensable for germinal center (GC) formation, but their mechanism of action is unclear. Here, we show by chromatin immunoprecipitation-sequencing that OBF1 extensively colocalizes with OCT1 and OCT2. We found that these factors also often colocalize with transcription factors of the ETS family. Furthermore, we showed that OBF1, OCT2, and OCT1 bind widely to the promoters or enhancers of genes involved in GC formation in mouse and human GC B cells. Short hairpin RNA knockdown experiments demonstrated that OCT1, OCT2, and OBF1 regulate each other and are essential for proliferation of GC-derived lymphoma cell lines. OBF1 downregulation disrupts the GC transcriptional program: genes involved in GC maintenance, such as BCL6, are downregulated, whereas genes related to exit from the GC program, such as IRF4, are upregulated. Ectopic expression of BCL6 does not restore the proliferation of GC-derived lymphoma cells depleted of OBF1 unless IRF4 is also depleted, indicating that OBF1 controls an essential regulatory node in GC differentiation.

Multi-color clonal tracking reveals intra-stage proliferative heterogeneity during mammary tumor progression.

Despite major progress in breast cancer research, the functional contribution of distinct cancer cell clones to malignant tumor progression and metastasis remains largely elusive. We have assessed clonal heterogeneity within individual primary tumors and metastases and also during the distinct stages of malignant tumor progression using clonal tracking of cancer cells in the MMTV-PyMT mouse model of metastatic breast cancer. Comparative gene expression analysis of clonal subpopulations reveals a substantial level of heterogeneity across and also within the various stages of breast carcinogenesis. The intra-stage heterogeneity is primarily manifested by differences in cell proliferation, also found within invasive carcinomas of luminal A-, luminal B-, and HER2-enriched human breast cancer. Surprisingly, in the mouse model of clonal tracing of cancer cells, chemotherapy mainly targets the slow-proliferative clonal populations and fails to efficiently repress the fast-proliferative populations. These insights may have considerable impact on therapy selection and response in breast cancer patients.

LATS1-Beclin1 mediates a non-canonical connection between the Hippo 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.

The cross-talk between the Hippo signaling pathway and autophagy:implications on physiology and cancer.

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.

Author Correction: PyMT-1099, a versatile murine cell model for EMT in breast cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Pygopus 2-Histone Interaction Is Critical for Cancer Cell Dedifferentiation and Progression in Malignant Breast Cancer.

Pygopus 2 (Pygo2) is a coactivator of Wnt/ß-catenin signaling that can bind bi- or trimethylated lysine 4 of histone-3 (H3K4me2/3) and participate in chromatin reading and writing. It remains unknown whether the Pygo2-H3K4me2/3 association has a functional relevance in breast cancer progression in vivo. To investigate the functional relevance of histone-binding activity of Pygo2 in malignant progression of breast cancer, we generated a knock-in mouse model where binding of Pygo2 to H3K4me2/3 was rendered ineffective. Loss of Pygo2-histone interaction resulted in smaller, differentiated, and less metastatic tumors, due, in part, to decreased canonical Wnt/ß-catenin signaling. RNA- and ATAC-sequencing analyses of tumor-derived cell lines revealed downregulation of TGFß signaling and upregulation of differentiation pathways such as PDGFR signaling. Increased differentiation correlated with a luminal cell fate that could be reversed by inhibition of PDGFR activity. Mechanistically, the Pygo2-histone interaction potentiated Wnt/ß-catenin signaling, in part, by repressing the expression of Wnt signaling antagonists. Furthermore, Pygo2 and ß-catenin regulated the expression of miR-29 family members, which, in turn, repressed PDGFR expression to promote dedifferentiation of wild-type Pygo2 mammary epithelial tumor cells. Collectively, these results demonstrate that the histone binding function of Pygo2 is important for driving dedifferentiation and malignancy of breast tumors, and loss of this binding activates various differentiation pathways that attenuate primary tumor growth and metastasis formation. Interfering with the Pygo2-H3K4me2/3 interaction may therefore serve as an attractive therapeutic target for metastatic breast cancer. SIGNIFICANCE: Pygo2 represents a potential therapeutic target in metastatic breast cancer, as its histone-binding capability promotes ß-catenin-mediated Wnt signaling and transcriptional control in breast cancer cell dedifferentiation, EMT, and metastasis.

A dual role of Irf1 in maintaining epithelial identity but also enabling EMT and metastasis formation of breast cancer cells.

An epithelial to mesenchymal transition (EMT) is an embryonic dedifferentiation program which is aberrantly activated in cancer cells to acquire cellular plasticity. This plasticity increases the ability of breast cancer cells to invade into surrounding tissue, to seed metastasis at distant sites and to resist to chemotherapy. In this study, we have observed a higher expression of interferon-related factors in basal-like and claudin-low subtypes of breast cancer in patients, known to be associated with EMT. Notably, Irf1 exerts essential functions during the EMT process, yet it is also required for the maintenance of an epithelial differentiation status of mammary gland epithelial cells: RNAi-mediated ablation of Irf1 in mammary epithelial cells results in the expression of mesenchymal factors and Smad transcriptional activity. Conversely, ablation of Irf1 during TGFß-induced EMT prevents a mesenchymal transition and stabilizes the expression of E-cadherin. In the basal-like murine breast cancer cell line 4T1, RNAi-mediated ablation of Irf1 reduces colony formation and cell migration in vitro and shedding of circulating tumor cells and metastasis formation in vivo. This context-dependent dual role of Irf1 in the regulation of epithelial-mesenchymal plasticity provides important new insights into the functional contribution and therapeutic potential of interferon-regulated factors in breast cancer.

Guidelines and definitions for research on epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by 'the EMT International Association' (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data generated in various experimental models and to promote cross-disciplinary collaboration to identify and address key open questions in this research field. While recognizing the importance of maintaining diversity in experimental approaches and conceptual frameworks, we emphasize that lasting contributions of EMT research to increasing our understanding of developmental processes and combatting cancer and other diseases depend on the adoption of a unified terminology to describe EMT.