Partial EMT Research Articles

Alpha-6 integrin deletion delays the formation of Brca1/p53-deficient basal-like breast tumors by restricting luminal progenitor cell expansion

BackgroundThe aberrant amplification of mammary luminal progenitors is at the origin of basal-like breast cancers associated with BRCA1 mutations. Integrins mediate cell–matrix adhesion and transmit mechanical and chemical signals that drive epithelial stem cell functions and regulate tumor progression, metastatic reactivation, and resistance to targeted therapies. Consistently, we have recently shown that laminin-binding integrins are essential for the expansion and differentiation of mammary luminal progenitors in physiological conditions. As over-expression of the laminin-binding α6 integrin (Itgα6) is associated with poor prognosis and reduced survival in breast cancer, we here investigate the role of Itgα6 in mammary tumorigenesis.MethodsWe used Blg-Cre; Brca1F/F; Trp53F/F mice, a model that phenocopies human basal-like breast cancer with BRCA1 mutations. We generated mutant mice proficient or deficient in Itgα6 expression and followed tumor formation. Mammary tumors and pretumoral tissues were characterized by immunohistochemistry, flow cytometry, RT-qPCR, Western blotting and organoid cultures. Clonogenicity of luminal progenitors from preneoplastic glands was studied in 3D Matrigel cultures.ResultsWe show that Itga6 deletion favors activation of p16 cell cycle inhibitor in the preneoplastic tissue. Subsequently, the amplification of luminal progenitors, the cell of origin of Brca1-deficient tumors, is restrained in Itgα6-deficient gland. In addition, the partial EMT program operating in Brca1/p53-deficient epithelium is attenuated in the absence of Itgα6. As a consequence of these events, mammary tumor formation is delayed in Itgα6-deficient mice. After tumor formation, the lack of Itgα6 does not affect tumor growth but rather alters their differentiation, resulting in reduced expression of basal cell markers.ConclusionsOur data indicate that Itgα6 has a pro-tumorigenic role in Blg-Cre; Brca1F/F; Trp53F/F mice developing basal-like mammary tumors. In particular, we reveal that Itgα6 is required for the luminal progenitor expansion and the aberrant partial EMT program that precedes the formation of BRCA1 deficient tumors.

Restoring BARX2 in OSCC reverses partial EMT and suppresses metastasis through miR-186-5p/miR-378a-3p-dependent SERPINE2 inhibition.

Tumor cells undergoing partial epithelial-mesenchymal transition (pEMT) are pivotal in local invasion and lymphatic metastasis of oral squamous cell carcinoma (OSCC), yet the mechanisms behind pEMT reversal remain poorly understood. In this study, the loss of BARX2 expression was revealed during the process of oral epithelial carcinogenesis and identified to activate the pEMT program, facilitate metastasis, and be associated with poor prognosis. Restoring BARX2 expression in OSCC cell lines effectively reversed tumor pEMT, evident in E/N-Cadherin switching, reduced cell invasion, proliferation, and stemness, and inhibited murine lung metastasis. BARX2 re-expression negatively correlated with several pEMT markers, notably SERPINE2, which was enriched in the invasive OSCC front, enhancing stemness and promoting metastasis, particularly in cervical lymph nodes. Furthermore, rescuing SERPINE2 impaired the inhibitory effect of BARX2 on the pEMT programs and reconstructed ECM through re-expression of MMP1. Mechanistically, we identified that BARX2 inhibited SERPINE2 through activating miR-186-5p and miR-378a-3p. These miRNAs, upregulated by BARX2, post-transcriptionally degraded SERPINE2 mRNA via targeting specific sequences. Blocking miR-186-5p and miR-378a-3p effectively abolished the negative regulatory effect of BARX2 on SERPINE2. Overall, our findings highlight BARX2 as a partial EMT-reverser in OSCC, providing fresh therapeutic prospects for restoring BARX2 signaling to inhibit invasion and metastasis.

Redox signalling regulates breast cancer metastasis via phenotypic and metabolic reprogramming due to p63 activation by HIF1α.

Redox signaling caused by knockdown (KD) of Glutathione Peroxidase 2 (GPx2) in the PyMT mammary tumour model promotes metastasis via phenotypic and metabolic reprogramming. However, the tumour cell subpopulations and transcriptional regulators governing these processes remained unknown. We used single-cell transcriptomics to decipher the tumour cell subpopulations stimulated by GPx2 KD in the PyMT mammary tumour and paired pulmonary metastases. We analyzed the EMT spectrum across the various tumour cell clusters using pseudotime trajectory analysis and elucidated the transcriptional and metabolic regulation of the hybrid EMT state. Integration of single-cell transcriptomics between the PyMT/GPx2 KD primary tumour and paired lung metastases unraveled a basal/mesenchymal-like cluster and several luminal-like clusters spanning an EMT spectrum. Interestingly, the luminal clusters at the primary tumour gained mesenchymal gene expression, resulting in epithelial/mesenchymal subpopulations fueled by oxidative phosphorylation (OXPHOS) and glycolysis. By contrast, at distant metastasis, the basal/mesenchymal-like cluster gained luminal and mesenchymal gene expression, resulting in a hybrid subpopulation using OXPHOS, supporting adaptive plasticity. Furthermore, p63 was dramatically upregulated in all hybrid clusters, implying a role in regulating partial EMT and MET at primary and distant sites, respectively. Importantly, these effects were reversed by HIF1α loss or GPx2 gain of function, resulting in metastasis suppression. Collectively, these results underscored a dramatic effect of redox signaling on p63 activation by HIF1α, underlying phenotypic and metabolic plasticity leading to mammary tumour metastasis.

Single-cell Analysis Reveals Inter- and Intratumour Heterogeneity in Metastatic Breast Cancer

Metastasis is the leading cause of cancer-related deaths of breast cancer patients. Some cancer cells in a tumour go through successive steps, referred to as the metastatic cascade, and give rise to metastases at a distant site. We know that the plasticity and heterogeneity of cancer cells play critical roles in metastasis but the precise underlying molecular mechanisms remain elusive. Here we aimed to identify molecular mechanisms of metastasis during colonization, one of the most important yet poorly understood steps of the cascade. We performed single-cell RNA-Seq (scRNA-Seq) on tumours and matched lung macrometastases of patient-derived xenografts of breast cancer. After correcting for confounding factors such as the cell cycle and the percentage of detected genes (PDG), we identified cells in three states in both tumours and metastases. Gene-set enrichment analysis revealed biological processes specific to proliferation and invasion in two states. Our findings suggest that these states are a balance between epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial transitions (MET) traits that results in so-called partial EMT phenotypes. Analysis of the top differentially expressed genes (DEGs) between these cell states revealed a common set of partial EMT transcription factors (TFs) controlling gene expression, including ZNF750, OVOL2, TP63, TFAP2C and HEY2. Our data suggest that the TFs related to EMT delineate different cell states in tumours and metastases. The results highlight the marked interpatient heterogeneity of breast cancer but identify common features of single cells from five models of metastatic breast cancer.

Gain-of-Function p53 Mutation Acts as a Genetic Switch for TGFβ Signaling-Induced Epithelial-to-Mesenchymal Transition in Intestinal Tumors.

KRAS and TP53 mutations shift activin-mediated signaling to overcome growth inhibition and promote partial EMT, identifying a subset of patients with colorectal cancer that could benefit from inhibition of TGFβ signaling.

Abstract PR06: Cooperative HPV and YAP-mediated oncogenic reprogramming of oral progenitors into cancer stem cells at single cell resolution

Abstract Human papillomavirus-positive head and neck squamous cell carcinoma (HPV+HNSC) constitutes the most common HPV-driven malignancy in the United States, and yet the molecular and cellular mechanisms underlying carcinogenesis in HPV+HNSC remain poorly understood. The decades long latency period and incomplete penetrance linking oral HPV infection to HPV+HNSC suggest that oncogenic events beyond E6-E7 expression are required for carcinogenesis. YAP is a transcriptional co-activator that coordinates cell stemness, growth, proliferation, and survival. We previously identified alterations resulting in YAP activation as a potential oncogenic event in HNSC. We thus hypothesized YAP activation in the context of E6-E7 expression may represent a key tumor initiating event leading to HPV+HNSC. We developed a genetically engineered murine system to investigate the effects of local induction of YAP activation and E6-E7-expression in Keratin14+ (K14+) oral epithelial progenitor cells (OEPCs). In this system, intralingual administration of tamoxifen and dietary administration of doxycycline enables spatiotemporally controlled expression of E6-E7 and/or YAP. Employing this model, we identify combined E6-E7 and YAP activation (E+Y) as sufficient to induce highly penetrant carcinoma formation as early as 10 days after transgene induction. Immunofluorescent analyses showed E+Y resulted in the ectopic expansion of a mitotically active (Ki67+), stem-like (TP63+, SOX2+) epithelial cell population with invasive potential. Bulk RNAseq of microdissected tongue epithelia showed that E+Y uniquely induced transcriptional programs promoting cell cycle progression, epithelial cell plasticity, and acute inflammatory response, which were not observed upon sole activation of either the E6-E7 or YAP transgene. scRNAseq of transgene-induced tongue epithelial cell suspensions identified 8 distinct clusters, including previously defined physiologic oral epithelial cell states and novel transgene-associated clusters. E+Y resulted in the emergence of a unique epithelial cell cluster with transcriptional characteristics consistent with a cancer stem cell population, with enrichment in YAP, mTORC1, E2F, and MYC-driven programs, and pan-cancer cell states including cell cycle, hypoxia, squamous differentiation, and partial EMT. Implantation of E+Y cells resulted in tumor formation in 100% of NSG mice, indicating that E+Y drives the emergence of a cancer stem cell population. We next defined transcriptionally co-expressed modules driving E+Y tumor initiating capacity. Among TCGA-HNSC subjects, 8 of 12 modules were enriched in tumor compared to matched normal tissue. Interestingly, 3 modules (G1/S, cell migration, and focal adhesion) were associated with overall and disease-free survival. Our findings show YAP activation in the context of E6-E7 expression is sufficient for rapid carcinogenesis. We illuminate cellular processes driving malignant conversion at single cell resolution, and show enrichment of these processes in human HNSC exhibiting poor prognosis. Citation Format: Farhoud Faraji, Sydney I. Ramirez, Lauren M. Clubb, Kuniaki Sato, Paola Y. Anguiano Quiroz, William M. G. Galloway, Thomas S. Hoang, Kate Medetgul-Ernar, Pauline Marangoni, Alfredo A. Molinolo, Joseph A. Califano, Quinton Smith, Ophir D. Klein, Pablo Tamayo, J. Silvio Gutkind. Cooperative HPV and YAP-mediated oncogenic reprogramming of oral progenitors into cancer stem cells at single cell resolution [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PR06.

Total RNA sequencing reveals gene expression and microbial alterations shared by oral pre-malignant lesions and cancer

Head and neck cancers are a complex malignancy comprising multiple anatomical sites, with cancer of the oral cavity ranking among the deadliest and the most disfiguring cancers globally. Oral cancer (OC) constitutes a subset of head and neck cancer cases, presenting primarily as tobacco- and alcohol-associated oral squamous cell carcinoma (OSCC), with a 5-year survival rate of ~ 65%, partly due to the lack of early detection and effective treatments. OSCC arises from premalignant lesions (PMLs) in the oral cavity through a multi-step series of clinical and histopathological stages, including varying degrees of epithelial dysplasia. To gain insights into the molecular mechanisms associated with the progression of PMLs to OSCC, we profiled the whole transcriptome of 66 human PMLs comprising leukoplakia with dysplasia and hyperkeratosis non-reactive (HkNR) pathologies, alongside healthy controls and OSCC. Our data revealed that PMLs were enriched in gene signatures associated with cellular plasticity, such as partial EMT (p-EMT) phenotypes, and with immune response. Integrated analyses of the host transcriptome and microbiome further highlighted a significant association between differential microbial abundance and PML pathway activity, suggesting a contribution of the oral microbiome toward PML evolution to OSCC. Collectively, this study reveals molecular processes associated with PML progression that may help early diagnosis and disease interception at an early stage.Graphical abstract

Abstract 4743: Wnt/beta-catenin mediated epigenetic modifications drive age-dependent oral squamous cell carcinoma evolution

Abstract Oral Squamous Cell Carcinoma (OSCC) is a devastating disease associated with high morbidity, poor survival, and few therapeutic options. This malignancy is driven, in part, by epigenetic reprogramming of the chromatin landscape directed by β-catenin in complex with CREB-binding protein (CBP) and mixed lineage leukemia methyltransferase 1 (MLL1). The β-catenin/CBP/MLL1 complex promotes an open chromatin structure by enabling transcription of genes associated with cell plasticity, including cancer stem cells and cells with partial EMT (p-EMT) phenotypes. Growing evidence indicates that cell plasticity and cellular senescence are integral processes shared by cancer and aging. Given that the median age of OSCC diagnosis is 66 years, it is likely that aging promotes OSCC evolution to advanced disease. To examine the effects of aging on OSCC evolution, we adapted a syngeneic mouse model of tobacco-associated oral carcinogenesis. This model utilizes a mouse cell line, 4MOSC1, derived from 4-nitroquinoline-1 oxide- (4NQO)-induced tongue tumor which, when implanted into mouse tongues, generates tumors that recapitulate human OSCC mutanome. The growth of 4MOSC1 derived orthotopic tumors was studied in 6- and 80-week-old mice. Tumors were allowed to develop for 18 days, after which they were harvested and processed for single cell RNA sequencing, histopathology and immunofluorescence (IF) analyses. Results showed that tumors grew at a faster rate and to a greater overall size in old mice compared to their young cohorts. OSCC harvested from the old mice displayed statistically significant reduction in membranous E-cadherin concomitant with increased Cbp and H3K4me3 abundance, suggesting an age-associated upregulation of β-catenin/Cbp/Mll1 epigenetic activity. Increased expression of Bmi1, keratin 14, and podoplanin supported increased cell plasticity in OSCC from aged mice. Further, these tumors exhibited augmented cellular senescence, as judged by the disruption of lamin B1-associated nuclear membrane integrity, and by an increased cell population with markers of senescence from single cell sequencing analyses. These data suggest that aging is associated with increased β-catenin/Cbp/Mll1 epigenetic signaling, cell plasticity and cellular senescence, which collectively contribute to the evolution of OSCC. Citation Format: Emily R. Fisher, Anthony Spinella, Nina C. Hardy, Xaralabos Varelas, Manish Bais, Maria A. Kukuruzinska. Wnt/beta-catenin mediated epigenetic modifications drive age-dependent oral squamous cell carcinoma evolution. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4743.

Single-copy Snail upregulation causes partial epithelial-mesenchymal transition in colon cancer cells

BackgroundEpithelial-mesenchymal transition (EMT) is an embryonic programme implicated in cancer stem cells, metastasis and therapeutic resistance. Its role in cancer progression remains controversial because the transition can be partial or complete in different models and contexts.MethodsUsing human colon cancer DLD-1 cells, we engineered a cell line with a single-copy of Snail that was doxycycline-inducible and compared it to existing EMT models in DLD-1. The effect of Snail upregulation was characterised functionally, morphologically, and by transcriptional profiling and protein expression.ResultsInduction with doxycycline increased Snail expression to a level similar to that observed in cancer cell lines spontaneously expressing Snail and results in partial EMT. In comparison, higher levels of overexpression arising from introduction of episomal-Snail, results in complete EMT. DLD-1 cells with partial EMT show chemoresistance in vitro, increased tumour growth in vivo and decreased apoptosis.ConclusionsThese findings highlight that the amount of bioavailable Snail can dictate phenotypic outcome and that partial EMT may be a preferred outcome of models operating within a natural range of Snail overexpression.

A multifunctional black phosphorus-based adhesive patch intrinsically induces partial EMT for effective burn wound healing.

The ultimate goal of cutaneous wound healing is to reform a stratified epithelium to restore the normal epidermal barrier, which involves the epithelial-to-mesenchymal transition (EMT) process. However, healing strategies based on EMT induction are immature and ambiguous to date. Excessive induction of EMT may cause fibrosis, hypertrophic scarring, and increased risk of malignancy. Here, we present a new EMT-inducing strategy for eliciting partial EMT to facilitate proper epithelial cell migration. The new EMT-inducing system integrates black phosphorus nanosheets (BPNSs), catechol-modified chitosan (CA-CS), and oxidized dextran (Odex) to engineer an adhesive hydrogel patch (C&BP-Patch) with remarkable efficacy on infectious burn wound healing. The C&BP-Patch can orchestrate key early skin wound healing processes including hemostasis, inflammation, and proliferation, which enable fast partial EMT induction to restore an intact epithelial barrier. The C&BP-Patch acts initially as a high-performance bio-sealant to create a moist and stable microenvironment for EMT. Moreover, the photothermal effects of the C&BP-Patch can eliminate bacteria, accelerate microcirculation and reduce inflammation to maintain a proper EMT. Most importantly, the BPNSs can intrinsically induce partial EMT of epithelial cells via a Snail1-mediated signaling pathway. Therefore, our study proposes a new strategy for effective infectious burn wound healing based on inducing partial EMT.

Protein Kinase D1 Signaling in Cancer Stem Cells with Epithelial-Mesenchymal Plasticity.

Pancreatic neuroendocrine tumors (pNETs) are extremely diverse and highly vascularized neoplasms that arise from endocrine cells in the pancreas. The pNETs harbor a subpopulation of stem cell-like malignant cells, known as cancer stem cells (CSCs), which contribute to intratumoral heterogeneity and promote tumor maintenance and recurrence. In this study, we demonstrate that CSCs in human pNETs co-express protein kinase PKD1 and CD44. We further identify PKD1 signaling as a critical pathway in the control of CSC maintenance in pNET cells. PKD1 signaling regulates the expression of a CSC- and EMT-related gene signature and promotes CSC self-renewal, likely leading to the preservation of a subpopulation of CSCs at an intermediate EMT state. This suggests that the PKD1 signaling pathway may be required for the development of a unique CSC phenotype with plasticity and partial EMT. Given that the signaling networks connected with CSC maintenance and EMT are complex, and extend through multiple levels of regulation, this study provides insight into signaling regulation of CSC plasticity and partial EMT in determining the fate of CSCs. Inhibition of the PKD1 pathway may facilitate the elimination of specific CSC subsets, thereby curbing tumor progression and metastasis.

Interplay between Partial EMT and Cisplatin Resistance as the Drivers for Recurrence in HNSCC.

This study aims to investigate the role of partial epithelial to mesenchymal transition (pEMT)-related proteins in modulating Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC). SCC-25 cells were pre-treated with TGF-beta1 followed by transient Krüppel-like Factor 4 (KLF4)-overexpression and Cisplatin treatment. Cell growth, cell morphological changes and cell migration were assessed using Juli BR live cell video-microscopy. In addition, Ki-67 and Slug immunostaining and follow-up image cytometric analysis of primary and recurrent HNSCC tumors were performed to evaluate the proliferation index (PI) and the EMT-like phenotype. We observed that proliferating and Slug-positive tumor cells expand after therapy in HNSCC. Subsequently, protein analysis revealed the stabilization of Slug, upregulation of Vimentin and phospho-p38 (p-p38) in Cisplatin-resistant SCC-25 cells. Moreover, KLF4-overexpression contributed to Cisplatin sensitivity by reduction of Slug at the protein level. This work strongly suggests that an pEMT-like pathway is activated in recurrent and Cisplatin-resistant HNSCC. Finally, stable KLF4-overexpression might sensitize HNSCC tumor cells for Cisplatin treatment.

Finding a Cytoskeletal Signature of Partial EMT Phenotype in Lung Cancer

Finding a Cytoskeletal Signature of Partial EMT Phenotype in Lung Cancer

Abstract 5368: Role of spatially distinct tumor fibroblast in erlotinib resistance

Abstract Metabolic reprogramming toward the hexosamine biosynthesis pathway (HBP), an understudied glucose pathway leading to O-glycosylation, is associated with poor survival in the lung adenocarcinoma (LUAD). We found that this metabolic switch happens primarily in Cancer-Associated Fibroblasts (CAFs), an “activated” phenotype associated with bad outcomes in cancer. We show that spatially distinct CAFs harvested from the tumor edge (Tumor-Adjacent-Fibroblasts; TAFs) of LUAD specimens vs the tumor core (Tumor Core Fibroblasts; TCFs) successfully protect cancer cells from erlotinib treatment, as measured by proliferation (ki67), migration (podoplanin) and resistance (Multidrug Resistance 1; MDR1) markers. However, while TCFs increase O-GlcNAcylation in cancer cells, a type of dynamic O-glycosylation, TAFs decrease O-GlcNAcylation in a subpopulation of cells. Interestingly, this subpopulation of cancer cells shows an increase of partial EMT features, as well as proliferation. In addition, we find this population of cancer cells to overexpress the MDR1 transporter, known to actively export erlotinib outside the cell. These results were reproduced in a novel in vitro assembloid model using patient-derived organoids and primary fibroblast mixtures that allow us to characterize cancer and spatially distinct stromal cells in response to erlotinib treatment. Overall, our preliminary results show that TAFs from the invasive leading edge of lung tumors decrease O-GlcNAcylation in cancer cells, which promotes erlotinib resistance. In summary, O-GlcNAcylation represents a potential avenue to mitigate resistance induced by tumor-stroma crosstalk and improve cancer treatment. Citation Format: Gina Bouchard, Sylvia K. Plevritis. Role of spatially distinct tumor fibroblast in erlotinib resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5368.

Abstract 2187: A multiplexed staining method to quantify EMT derived tumor heterogeneity presents a means of predicting patient prognosis based on EMT state

Abstract Intra-tumoral heterogeneity is a key contributor to poor prognosis and metastatic disease in breast cancer. This study seeks to detect and quantify intra-tumoral heterogeneity deriving from the Epithelial-to-Mesenchymal transition using a multi-round, multiplexed immunofluorescence staining approach to delineate EMT states at a single cell resolution in primary tumor specimens to aid in pathological patient diagnosis. Despite major advances in our understanding, the contributions of EMT research to improvements in diagnostic pathology or cancer therapy have been minimal. One reason for this gap stems from our inability to accurately detect and quantify epithelial-mesenchymal heterogeneity in primary tumor specimens. Secondly, the significance of recently identified intermediate or partial EMT states to predicting tumor prognosis and therapy response are unclear. To study the role of various states within the EMT spectrum and their regulatory networks, the heterogeneous breast cancer cell line, SUM149PT, was used to derive six single cell clones encompassing the spectrum of EMT states, from epithelial to mesenchymal. Interrogation of this model system in vivo has revealed increased tumor growth and metastatic potential in the intermediate EMT states when compared to the extreme epithelial and mesenchymal states. To further elucidate EMT states in vivo, we employ a 6-marker multi-round immunofluorescence-based staining approach to identify cells that reside in various states along the EMT spectrum. We subsequently used cell-segmented, entropy-based approach and weighted phenotype analysis on these tumors with the purpose of scoring heterogeneity and overall EMT state. Notably, this analysis segregated stromal infiltrates and their contributions to aggregate EMT scoring, which has been a major hurdle in using EMT as a scoring metric in patient samples. Overall, SUM149 clone-derived tumors held true to the relative EMT states of the starting cell populations; intermediate-derived tumors displayed high heterogeneity while epithelial and mesenchymal clone-derived tumors had lower levels of heterogeneity, despite retaining different EMT scores. Decoupling of heterogeneity and EMT state in this way provides two metrics to assess potential metastatic ability of a tumor. This staining method and analysis has been successfully applied in a preliminary set of patient tumors, showing promise for these two factors, E-M Heterogeneity and EMT score, as a tumor prognostic indicator to inform therapeutic decision-making. In the future, this staining and quantification shows promise as a means of predicting patient prognosis and informing potential treatment options based on targeting EMT states. Citation Format: Meredith S. Brown. A multiplexed staining method to quantify EMT derived tumor heterogeneity presents a means of predicting patient prognosis based on EMT state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2187.

Invadopodia play a role in prostate cancer progression

BackgroundInvadopodia, actin-rich structures that release metallo-proteases at the interface with extra-cellular matrix, in a punctate manner are thought to be important drivers of tumour invasion. Invadopodia formation has been observed in-vitro and in-vivo in numerous metastatic cell lines derived from multiple tumour types. However, prostate cancer cell lines have not been routinely reported to generate invadopodia and the few instances have always required external stimulation.MethodsIn this study, the invasive potential of primary prostate adenocarcinoma cell lines, which have never been fully characterised before, was investigated both in-vitro invadopodia assays and in-vivo zebrafish dissemination assay. Subsequently, circulating tumour cells from prostate cancer patients were isolated and tested in the invadopodia assay.ResultsRetention of E-cadherin and N-cadherin expression indicated a transitional state of EMT progression, consistent with the idea of partial EMT that has been frequently observed in aggressive prostate cancer. All cell lines tested were capable of spontaneous invadopodia formation and possess a significant degradative ability in-vitro under basal conditions. These cell lines were invasive in-vivo and produced visible metastasis in the zebrafish dissemination assay. Importantly we have proceeded to demonstrate that circulating tumour cells isolated from prostate cancer patients exhibit invadopodia-like structures and degrade matrix with visible puncta. This work supports a role for invadopodia activity as one of the mechanisms of dissemination employed by prostate cancer cells.ConclusionThe combination of studies presented here provide clear evidence that invadopodia activity can play a role in prostate cancer progression.

Abstract P3-09-12: Breast cancer derived GATA3 mutations disrupt luminal transcriptional network

Abstract GATA3 has been identified as one of the most frequently mutated genes in breast cancer. In the METABRIC cohort, among 1,980 patient cases, 230 breast cancers harbored GATA3 mutations (~12%). 75% of the GATA mutations were observed in luminal breast tumors (47% in luminal A, 28% in luminal B). The recent genomic data in metastatic breast cancer also showed that the frequency of GATA3 somatic mutations was even higher in the metastatic breast cancer cohort. Lung, lymph nodes, and brain metastases were observed in the GATA3 mutant breast cancer patients. Based on these patient genomic data, GATA3 mutations have been considered as cancer drivers, yet the functional consequences of GATA3 mutations are underexplored. We and other groups previously identified that patients carrying GATA3 mutations have diverse clinical features. More than 70% of cases are small nucleotide deletions or insertions (indel), while less than 30% are missense mutations. By classifying the GATA3 indel mutations into 4 groups, we observed distinct clinical features. Somatic mutations found in the GATA3 second zinc-finger domain (ZnFn2) are significantly associated with poorer patient outcomes including worse patient survival. ZnFn2 mutations are predominantly found in luminal B breast tumors, while splice site mutations are frequently found in luminal A breast tumors and associated with better patient survival. These distinct clinical features clearly suggest the differential impacts of GATA3 mutations on breast cancer cells. To dissect the function of GATA3 mutants, we developed a GATA3 mutant cell line (R330fs/WT T47D cells) by CRISPR, which endogenously expresses a heterozygous R330 frame-shift (R330fs) mutant. R330fs mutations are found in multiple data cohorts. The majority (>90%) of GATA3 mutations are heterozygous. Therefore, this R330fs mutant T47D cell line mimics the type of alteration found in patients. Importantly, we found that the R330fs mutation induces transcriptional reprogramming in T47D luminal breast cancer cells leading to more aggressive phenotypes such as faster tumor growth. In the mutant cells, many epithelial marker genes (such as progesterone receptor and TFF1) were down-regulated while mesenchymal marker genes (such as TWIST1 and SNAI2) are up-regulated suggesting that R330fs induces a partial EMT in T47D cells. We also found that transcriptional changes in the R330fs mutant cells were strongly associated with redistribution of GATA3, Estrogen Receptor alpha, and FOXA1. Gene expression profiles in the other GATA3 mutant breast cancer cell lines also suggest similar genomic alterations. These results suggest the active roles of GATA3 mutations during breast cancer development. Citation Format: Motoki Takaku, Mika Saotome, Renju Nair. Breast cancer derived GATA3 mutations disrupt luminal transcriptional network [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-12.

Canonical TGF\u03b2 signaling induces collective invasion in colorectal carcinogenesis through a Snail1- and Zeb1-independent partial EMT

Local invasion is the initial step towards metastasis, the main cause of cancer mortality. In human colorectal cancer (CRC), malignant cells predominantly invade as cohesive collectives and may undergo partial epithelial-mesenchymal transition (pEMT) at the invasive front. How this particular mode of stromal infiltration is generated is unknown. Here we investigated the impact of oncogenic transformation and the microenvironment on tumor cell invasion using genetically engineered organoids as CRC models. We found that inactivation of the Apc tumor suppressor combined with expression of oncogenic KrasG12D and dominant-negative Trp53R172H did not cell-autonomously induce invasion in vitro. However, oncogenic transformation primed organoids for activation of a collective invasion program upon exposure to the prototypical microenvironmental factor TGFβ1. Execution of this program co-depended on a permissive extracellular matrix which was further actively remodeled by invading organoids. Although organoids shed some epithelial properties particularly at the invasive edge, TGFβ1-stimulated organoids largely maintained epithelial gene expression while additionally implementing a mesenchymal transcription pattern, resulting in a pEMT phenotype that did not progress to a fully mesenchymal state. Notably, while TGFβ1 induced pEMT and promoted collective invasion, it abrogated self-renewal capacity of TKA organoids which correlated with the downregulation of intestinal stem cell (ISC) marker genes. Mechanistically, induction of the non-progressive pEMT required canonical TGFβ signaling mediated by Smad transcription factors (TFs), whereas the EMT master regulators Snail1 and Zeb1 were dispensable. Gene expression profiling provided further evidence for pEMT of TGFβ1-treated organoids and showed that their transcriptomes resemble those of human poor prognosis CMS4 cancers which likewise exhibit pEMT features. We propose that collective invasion in colorectal carcinogenesis is triggered by microenvironmental stimuli through activation of a novel, transcription-mediated form of non-progressive pEMT independently of classical EMT regulators.

Therapeutically Targeting Cancers That Overexpress FOXC1: A Transcriptional Driver of Cell Plasticity, Partial EMT, and Cancer Metastasis.

Metastasis accounts for more than 90% of cancer related mortality, thus the most pressing need in the field of oncology today is the ability to accurately predict future onset of metastatic disease, ideally at the time of initial diagnosis. As opposed to current practice, what would be desirable is that prognostic, biomarker-based detection of metastatic propensity and heightened risk of cancer recurrence be performed long before overt metastasis has set in. Without such timely information it will be impossible to formulate a rational therapeutic treatment plan to favorably alter the trajectory of disease progression. In order to help inform rational selection of targeted therapeutics, any recurrence/metastasis risk prediction strategy must occur with the paired identification of novel prognostic biomarkers and their underlying molecular regulatory mechanisms that help drive cancer recurrence/metastasis (i.e. recurrence biomarkers). Traditional clinical factors alone (such as TNM staging criteria) are no longer adequately prognostic for this purpose in the current molecular era. FOXC1 is a pivotal transcription factor that has been functionally implicated to drive cancer metastasis and has been demonstrated to be an independent predictor of heightened metastatic risk, at the time of initial diagnosis. In this review, we present our viewpoints on the master regulatory role that FOXC1 plays in mediating cancer stem cell traits that include cellular plasticity, partial EMT, treatment resistance, cancer invasion and cancer migration during cancer progression and metastasis. We also highlight potential therapeutic strategies to target cancers that are, or have evolved to become, “transcriptionally addicted” to FOXC1. The potential role of FOXC1 expression status in predicting the efficacy of these identified therapeutic approaches merits evaluation in clinical trials.

Abstract LB242: Activation of androgen receptor induces partial epithelial-mesenchymal transition of breast MDA-MB-453 cancer cells: Alterations of a molecular network

Abstract Epithelial-mesenchymal transition (EMT) of tumor cells is prerequisite to cancer cell invasion and metastasis. This process involves an intricate molecular network. Androgen receptor (AR) appears to play an important role in the biology of breast cancers, particularly those dependent on AR expression such as luminal AR (LAR) breast cancer subtype. We and others have previously shown that treatment of LAR breast MDA-MB-453 cancer cells with dihydrotestosterone (DHT) induces a mesenchymal phenotype via AR. In this study, the cellular and molecular dynamics of DHT-induced EMT in MDA-MB-453 cells were investigated. A plethora of techniques were used including PCR arrays, immunoblotting, immunoprecipitation, immunofluorescence, cell fractionation, and siRNA transfection. Change of cell morphology was concomitant with increased cell migration after treating cells with DHT. Exposure of cells to DHT for one hour was sufficient to induce changes in cell morphology and actin cytoskeleton after 72 hours indicating altered gene expression. Investigating changes in the expression of 84 EMT-related genes revealed induced expression of the transcription factor Slug and down-expression of the desmosomal DSC2, but, surprisingly DHT also reduced levels of pro-EMT β-catenin, N-cadherin, and TCF-4, and increased expression of anti-EMT RGS2, all of which were confirmed at the protein level. Not only early interaction of AR and β-catenin was observed following AR activation, inhibition of β-catenin blocked DHT-induced mesenchymal transition of cells and their migration. Interestingly, inhibition of AR prevented mesenchymal transition of cells by activation of β-catenin suggesting reciprocal requirement of both molecules. Activation of AR also resulted in its nuclear translocation and interaction with Slug. Knocking down Slug blocked DHT-induced cellular changes and also reduced basal, but not DHT-induced, cell migration indicating that mesenchymal transition of these cells and their migration are independent processes. Furthermore, not only activation of AR lowered expression and altered the pattern of and spatial intracellular localization of DSC2, the decrease in DSC2 levels was sufficient to induce EMT-like features in a β-catenin-dependent, but AR-independent manner. Expression of DSC2 was under the negative regulation of Slug. Shortly after AR activation, AR-RGS2 interaction increased concomitant with a significant increase of RGS2 protein without a change in its cellular localization. Transfection of cells with RGS2 siRNA was sufficient to induce mesenchymal characteristics of cells even in the absence of DHT. Interestingly, this morphological transition was blocked by inhibiting AR. These results indicate that AR modulates a molecular network in inducing partial EMT of MDA-MB-453 cells. Citation Format: Mamoun Ahram, Randa Bawadi, Mohammad Abdullah, Dana Alsafadi, Shahd Arqam. Activation of androgen receptor induces partial epithelial-mesenchymal transition of breast MDA-MB-453 cancer cells: Alterations of a molecular network [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB242.