Mesenchymal-like Cancer Cells Research Articles

PKN2 is a dependency of the mesenchymal-like cancer cell state.

Cancer cells exploit a mesenchymal-like transcriptional state (MLS) to survive drug treatments. Although the MLS is well characterized, few therapeutic vulnerabilities targeting this program have been identified. Here, we systematically identify the dependency network of mesenchymal-like cancers through an analysis of gene essentiality scores in ~800 cancer cell lines, nominating a poorly studied kinase, PKN2, as a top therapeutic target of the MLS. Co-essentiality relationships, biochemical experiments, and genomic analyses of patient tumors revealed that PKN2 promotes mesenchymal-like cancer growth through a PKN2-SAV1-TAZ signaling mechanism. Notably, pairing genetic PKN2 inhibition with clinically relevant targeted therapies against EGFR, KRAS, and BRAF oncogenes suppresses drug resistance by depleting mesenchymal-like drug-tolerant persister cells. These findings provide evidence that PKN2 is a core regulator of the Hippo tumor suppressor pathway and highlight the potential of PKN2 inhibition as a generalizable therapeutic strategy to overcome drug resistance driven by the MLS across cancer contexts.

The biotoxin BMAA promotes mesenchymal transition in neuroblastoma cells.

Mesenchymal-like cancer cells are an indicator of malignant tumors as they exhibit tumorigenic properties including downregulation of differentiation markers, and increased colony-forming potential, motility, and chemoresistance. We have previously demonstrated that the cyanobacterial biotoxin beta-methylamino-L-alanine (BMAA) is capable of influencing neural cell differentiation state through mechanisms involving the Wnt signaling pathway, suggesting the possibility that BMAA may play a role in influencing other Wnt related differentiation processes including mesenchymal transition. In this study we present evidence characterizing the effects of BMAA on mesenchymal transition in a human neuroblastoma cell line and provide support for the hypothesis that the biotoxin can promote this process in these cells by altering differentiation state, inducing changes in gene expression, and changing cellular function in manners consistent with cellular mesenchymal transition. Results of this study indicate that BMAA exposure may promote carcinogenesis through its effects on cell differentiation state in certain contexts. These results suggest that exposure to the biotoxin BMAA may be an influencing factor in chemotherapy resistance and cancer relapse in neuroblastoma.

A Genuinely Hybrid, Multiscale 3D Cancer Invasion and Metastasis Modelling Framework

We introduce in this paper substantial enhancements to a previously proposed hybrid multiscale cancer invasion modelling framework to better reflect the biological reality and dynamics of cancer. These model updates contribute to a more accurate representation of cancer dynamics, they provide deeper insights and enhance our predictive capabilities. Key updates include the integration of porous medium-like diffusion for the evolution of Epithelial-like Cancer Cells and other essential cellular constituents of the system, more realistic modelling of Epithelial–Mesenchymal Transition and Mesenchymal–Epithelial Transition models with the inclusion of Transforming Growth Factor beta within the tumour microenvironment, and the introduction of Compound Poisson Process in the Stochastic Differential Equations that describe the migration behaviour of the Mesenchymal-like Cancer Cells. Another innovative feature of the model is its extension into a multi-organ metastatic framework. This framework connects various organs through a circulatory network, enabling the study of how cancer cells spread to secondary sites.

Abstract 274: Cancer-associated fibroblasts exhibit increased motility under hypoxic conditions via interaction with cancer cells

Abstract Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment, playing various roles through interactions with cancer cells and other stromal cell types. In a 3D co-culture system of CAFs and lung cancer cells, a specific subgroup of CAFs exhibited increased motility, while another subgroup of CAFs remained stationary. Using the photoconvertible fluorescent protein Dendra2, we isolated these distinct CAF subgroups and conducted RNA sequencing. Gene set enrichment and gene ontology analysis revealed that upregulated genes in motile CAFs are associated with hypoxia and glycolysis. Conditioned medium from mesenchymal-like cancer cells enhanced the expression of genes related to hypoxia and glycolysis in CAFs. Among these genes, Aldoa and Ldha were chosen for knockdown in CAFs, resulting in a reduction in CAF motility. We confirmed that mesenchymal-like cancer cells promoted CAF motility and intensified hypoxic conditions within the co-culture system, which was monitored using a hypoxia reporter. Ceruloplasmin (CP) was identified as a secretory protein from mesenchymal-like cancer cells through mass spectrometry. Knockdown of CP led to the inhibition of CAF motility. In conclusion, the motility of CAFs is enhanced under hypoxic conditions facilitated by the secretion of CP from mesenchymal-like cancer cells in the lung tumor microenvironment. Citation Format: Jihye Park, Sieun Lee, Jonathan M. Kurie, Young-Ho Ahn. Cancer-associated fibroblasts exhibit increased motility under hypoxic conditions via interaction with cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 274.

Epithelial and Mesenchymal-like Pancreatic Cancer Cells Exhibit Different Stem Cell Phenotypes Associated with Different Metastatic Propensities.

Pancreatic ductal adenocarcinoma (PDAC) is mostly diagnosed at advanced or even metastasized stages, limiting the prognoses of patients. Metastasis requires high tumor cell plasticity, implying phenotypic switching in response to changing environments. Here, epithelial-mesenchymal transition (EMT), being associated with an increase in cancer stem cell (CSC) properties, and its reversion are important. Since it is poorly understood whether different CSC phenotypes exist along the EMT axis and how these impact malignancy-associated properties, we aimed to characterize CSC populations of epithelial and mesenchymal-like PDAC cells. Single-cell cloning revealed CSC (Holoclone) and non-CSC (Paraclone) clones from the PDAC cell lines Panc1 and Panc89. The Panc1 Holoclone cells showed a mesenchymal-like phenotype, dominated by a high expression of the stemness marker Nestin, while the Panc89 Holoclone cells exhibited a SOX2-dominated epithelial phenotype. The Panc89 Holoclone cells showed enhanced cell growth and a self-renewal capacity but slow cluster-like invasion. Contrarily, the Panc1 Holoclone cells showed slower cell growth and self-renewal ability but were highly invasive. Moreover, cell variants differentially responded to chemotherapy. In vivo, the Panc1 and Panc89 cell variants significantly differed regarding the number and size of metastases, as well as organ manifestation, leading to different survival outcomes. Overall, these data support the existence of different CSC phenotypes along the EMT axis in PDAC, manifesting different metastatic propensities.

Regulating POLR3G by MicroRNA-26a-5p as a promising therapeutic target of lung cancer stemness and chemosensitivity.

Cancer stem cells (CSCs) identified in lung cancer exhibit resistance to chemotherapy, radiotherapy, and targeted therapy. Therefore, a technology for controlling CSCs is needed to overcome such resistance to cancer therapy. Various evidences about the association between epithelial-mesenchymal transition related transcriptomic alteration and acquisition of CSC phenotype have been proposed recently. Down-regulated miR-26a-5p is closely related to mesenchymal-like lung cancer cell lines. These findings suggest that miR-26a-5p might be involved in lung cancer stemness. RNA polymerase III subunit G (POLR3G) was selected as a candidate target of miR-26a-5p related to cancer stemness. It was found that miR-26a-5p directly regulates the expression of POLR3G.Overexpression of miR-26a-5p induced a marked reduction of colony formation and sphere formation. Co-treatment of miR-26a-5p and paclitaxel decreased cell growth, suggesting that miR-26a-5p might play a role as a chemotherapy sensitizer. In the cancer genome atlas data, high miR-26a-5p and low POLR3G expression were also related to higher survival rate of patients with lung adenocarcinoma. These results suggest that miR-26a-5p can suppress lung cancer stemness and make cancer cell become sensitive to chemotherapy. This finding provides a novel insight into a potential lung cancer treatment by regulating stemness.

Abstract 1342: Roles of mesenchymal-like tumour cells and myofibroblastic cancer-associated fibroblasts in progression and therapeutic response of clear-cell renal cell carcinoma

Abstract Purpose: Immune checkpoint inhibitors (ICI) represent the cornerstone for treatment of patients with metastatic clear-cell renal cell carcinoma (ccRCC). Despite a favourable response for a subset of patients, others present primary progressive disease highlighting the need to better understand plasticity of cancer cells and their crosstalk with the microenvironment to better predict therapeutic response and personalize treatment. Experimental design: We performed single-cell RNA sequencing on 56,421 cells from tumour and normal adjacent tissue of patients with ccRCC at different disease stages. Identification of cancer and microenvironment cell populations was validated by deconvolution analyses of public datasets and their localizations within tumours were determined by spatial transcriptomic analyses. We also assessed association of tumour and microenvironment populations with response of ICI treated patients using data from the BIONIKK clinical trial (NCT02960906). Results: We identify 46 cell populations including 5 tumour subpopulations characterized by distinct transcriptional signatures representing an epithelial to mesenchymal transition gradient as well as novel inflamed state. Deconvolution of the tumour and microenvironment signatures revealed strong correlation between mesenchymal-like ccRCC cancer cells and myofibroblastic cancer-associated fibroblasts (myCAFs) that associate with metastasis and tumour aggressiveness. Spatial transcriptomics revealed their co-localization and we identified therapeutically targetable ligand-receptor interactions underlying their crosstalk. Finally, we show that enrichment in myCAFs was associated with primary resistance to ICI therapy in the BIONIKK clinical trial. Conclusions: We describe epithelial-mesenchymal plasticity of ccRCC cancer cells and identify potentially targetable pathways involved in tumour cell-myCAFs crosstalk opening the way for personalized treatment of ccRCC patients. Citation Format: Alexandra Helleux, Guillaume Davidson, Yann A. Vano, Véronique Lindner, Antonin Fattori, Marie Cerciat, Reza T. Elaidi, Virginie Verkarre, Cheng-Ming Sun, Christine Chevreau, Mostefa Bennamoun, Hervé Lang, Thibault Tricard, Wolf H. Fridman, Catherine Sautes-Fridman, Xiaoping Su, Damien Plassard, Céline Keime, Christelle Thibault-Carpentier, Philippe Barthelemy, Stéphane Oudard, Irwin Davidson, Gabriel G. Malouf. Roles of mesenchymal-like tumour cells and myofibroblastic cancer-associated fibroblasts in progression and therapeutic response of clear-cell renal cell carcinoma [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 1342.

YBX1 integration of oncogenic PI3K/mTOR signalling regulates the fitness of malignant epithelial cells

In heterogeneous head and neck cancer (HNC), subtype-specific treatment regimens are currently missing. An integrated analysis of patient HNC subtypes using single-cell sequencing and proteome profiles reveals an epithelial-mesenchymal transition (EMT) signature within the epithelial cancer-cell population. The EMT signature coincides with PI3K/mTOR inactivation in the mesenchymal subtype. Conversely, the signature is suppressed in epithelial cells of the basal subtype which exhibits hyperactive PI3K/mTOR signalling. We further identify YBX1 phosphorylation, downstream of the PI3K/mTOR pathway, restraining basal-like cancer cell proliferation. In contrast, YBX1 acts as a safeguard against the proliferation-to-invasion switch in mesenchymal-like epithelial cancer cells, and its loss accentuates partial-EMT and in vivo invasion. Interestingly, phospho-YBX1 that is mutually exclusive to partial-EMT, emerges as a prognostic marker for overall patient outcomes. These findings create a unique opportunity to sensitise mesenchymal cancer cells to PI3K/mTOR inhibitors by shifting them towards a basal-like subtype as a promising therapeutic approach against HNC.

1722P The subtype-specific cell fitness of head and neck cancer is regulated by the YBX1 integration of oncogenic PI3K/mTOR signalling

Molecular subtyping of heterogeneous cancers has gained substantial biological interest although with limited translation to the clinic. In heterogeneous head and neck cancer (HNC), subtype-specific treatment regimens are currently missing. A multi-omic approach integrating whole-genome and single-cell transcriptome sequencing with reverse-phase protein array was employed in patient primary and metastatic tumours and human cell lines to investigate the subtype-specific molecular mechanisms as the therapeutic vulnerabilities in HNC. An integrated analysis of HNC subtypes using single-cell sequencing and proteome profiles revealed a hallmark epithelial-mesenchymal transition (EMT) signature within the epithelial cancer-cell population. The EMT signature was evident in the invasive mesenchymal subtype and surprisingly coincided with PI3K/mTOR pathway inactivation. Conversely, the EMT signature was suppressed in epithelial cancer cells of the basal subtype which exhibited hyperactive PI3K/mTOR oncogenic signalling. We further identified that phosphorylation of Y Box binding protein 1 (YBX1), downstream of the PI3K/mTOR pathway, restrained the basal-like cancer cell proliferation. In contrast, YBX1 acted as a safeguard against the proliferation to invasion switch in mesenchymal-like epithelial cancer cells, and its loss accentuated partial EMT acquisition and in vivo invasion. Interestingly, the proteomic analysis of patient HNC identified phospho-YBX1 as a prognostic marker for overall patient outcomes, the loss of which correlated with HNC metastasis and poor prognosis. Our results indicate that HNC cells adopt a PI3K-phospho-YBX1-dependent proliferation program that is mutually exclusive to partial EMT involvement which relies on the lack of phosphorylation of YBX1 in PI3K-inactive cancer cells. These findings create a unique opportunity to sensitise mesenchymal cancer cells to PI3K/mTOR inhibitors by shifting them towards a basal-like subtype, constituting a promising therapeutic approach against HNC.

Abstract SY31-01: Tissue resident macrophages and a mesenchymal-like program control early disseminated cancer cell dormancy

Abstract Tissue resident macrophages and a mesenchymal-like program control early disseminated cancer cell dormancy. Julio A. Aguirre-GhisoDepartment of Cell Biology, Department of Medicine-Oncology, Cancer Dormancy and Tumor Microenvironment Institute, Albert Einstein Cancer Center, Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Increasing evidence shows that cancer cells can disseminate from early-evolved primary lesions much earlier than the classical metastasis models predicted. The current paradigm supports that the tissue microenvironment where disseminated cancer cells (DCCs) lodge is a critical determinant of the timing of metastasis. However, how tissue-resident myeloid cells control this process is largely unclear. The lung tissue contains a resident population of alveolar macrophages (AMs), whose function in metastatic dormancy and growth is largely unexplored. Here, we used various transgenic mouse models, human sample analysis and single cell RNA-sequencing to reveal DCC heterogeneity and plasticity in the lung across disease evolution. We found a previously unrecognized role of mesenchymal- and pluripotency-like (M-like) programs driven by the transcription factor ZFP281 in coordinating early cancer cell spread and a long-lived dormancy program in early DCCs. The gene profiles revealed molecules that would allow early DCCs to crosstalk with AMs. Using a 3D Matrigel assay, we found that AMs are responsible for inducing an M-like phenotype in cancer cells (of both early and late evolved cancer cells) and exclusively suppressed growth of early lesion (EL) HER2+ cancer cells. The conditioned media (CM) from AMs was also found to induce an M-like phenotype and growth arrest of EL cells. RNA-sequencing profiling of pure EL cells and AMs that had been co-cultured revealed a reciprocal influence of the two cell types: increased ECM organization and induction of invasiveness in EL cells, as well as changes in inflammatory response and immune programs in AMs. Prediction of ligand-receptor interactions using the NATMI tool allowed for identification of signaling axis that would allow AMs to induce an M-like and growth program in EL cells. To test whether indeed AMs are inducing an M-like and growth arrest phenotype in early DCCs in lungs, we depleted AMs in mice bearing early MMTV-HER2 or late evolved E0771 breast cancer cells in lungs via intranasal instillation of clodronate liposomes or via the use of a AM=specific CD169-DTR targeting system. Following AM depletion, dormant DCCs began to proliferate and formed clusters and overt metastases. Our results support that tissue resident macrophages play a key role in limiting metastatic expansion of breast cancer cells via the induction of a DCC mesenchymal-like dormancy program. Citation Format: Julio A. Aguirre-Ghiso. Tissue resident macrophages and a mesenchymal-like program control early disseminated cancer cell dormancy [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 SY31-01.

Abstract 817: microRNA-26a-5p is a prognostic factor that regulates cancer stemness

Abstract Background: Cancer stem cells (CSCs) identified in lung cancer exhibit resistance to chemotherapy, radiotherapy, and targeted therapy. Therefore, a technology to control of CSCs is needed to overcome such resistance to cancer therapy. Various evidences about the association between epithelial-mesenchymal transition related transcriptomic alteration and acquisition of CSC phenotype have been proposed recently. In our previous research, down-regulated miR-26a-5p is closely related to mesenchymal-like lung cancer cell lines. These findings suggest that miR-26a-5p might be involved in lung cancer stemness. Methods: RNA polymerase III subunit G (POLR3G) was selected as a candidate target of miR-26a-5p related to cancer stemness. its quantitative relationship was investigated by polymerase chain reaction, western blot after transfection of miR-26a-5p. luciferase assay were done for investigating the direct regulation of miR-26a-5p on POLR3G expression. After transfection of miR-26a-5p, colony formation assay and sphere formation assay were performed to evaluate the effect on cancer stemness. By treating cancer cell by miR-26a-5p and paclitaxel, cell viability was checked by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and Muse cell analyzer. Expression level of each gene and its impact on survival were revealed by the cancer genome atlas pancancer database. Results: miR-26a-5p regulated the expression of POLR3G directly. Overexpression of miR-26a-5p induced down regulation of POLR3G and a marked reduction of colony formation and sphere formation. Co-treatment of miR-26a-5p with paclitaxel decreased cell growth, suggesting that miR-26a-5p might play a role as a chemotherapy sensitizer. In the cancer genome atlas data, down-regulated miR-26a-5p and up-regulated POLR3G were shown compared to adjacent normal tissue. High miR-26a-5p and low POLR3G expression were also related to higher survival rate of patients with lung adenocarcinoma. Conclusions: Overexpression of miR-26a-5p can suppress lung cancer stemness and make cancer cell become sensitive to chemotherapy. This finding provides a novel insight into a potential lung cancer treatment by regulating stemness. Citation Format: Daeun Kang, Chang Ryul Park, Minhyeok Lee, Su Yel Lee, Se Jin Park, Wan Jin Hwang, Gwan Woo Ku, Seong Lan Yu, In Beom Jeong, Sun Jung Kwon, Jaeku Kang, Eung Bae Lee, Ji Woong Son. microRNA-26a-5p is a prognostic factor that regulates cancer stemness [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 817.

Abstract 2989: FAP facilitates ovary tumorigenesis through the NF-κB/survivin funcational axis

Abstract Ovarian cancer is the deadliest gynecologic cancer, mainly attributing to lack of early diagnosis of the diseases. Current mainstay treatment for ovarian cancer is surgical removal followed by chemotherapy. Although patients initially respond well, most of patients suffer from relapse and die from metastasis. Therefore, developing successful approaches combatting metastasis is most likely to prolong patients’ survival and improve their life quality. Toward this goal, we analyzed The Cancer Genome Atlas ovarian cancer patient dataset to reach for targetable genes preferentially elevated in advanced ovarian cancer. Among them, we found that knockdown of fibroblast activation protein alpha (FAP) led to dramatic apoptosis in mesenchymal-like ovarian cancer cells. To elucidate the underlying mechanism, we performed RNAseq and identified that TNF-alpha/NF-κB signaling pathway was impaired in FAP-knockdown cells. NF-κB signaling pathway is well characterized to promote cell survival by elevating the expression of pro-survival genes. In fact, the level of survivin was diminished upon FAP-knockdown. As forced expression of survivin almost completely abrogated apoptosis elicited by FAP-knockdown and survivin inhibitor also induced robust apoptosis in ovarian cancer cells, we reason that FAP participates in ovarian cancer development by promoting cell survival through activation of NF-κB/survivin axis. With the aid of EpCAM aptamer as delivering cargo, we demonstrated that FAP siRNA was able to suppress intraperitoneal ovarian cancer development. This study suggests that FAP is an ideal therapeutic target against mesenchymal-like ovarian cancer. Citation Format: Bin Li, Shuang Huang. FAP facilitates ovary tumorigenesis through the NF-κB/survivin funcational axis [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 2989.

Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models.

The lack of inadequate preclinical models remains a limitation for cancer drug development and is a primary contributor to anti-cancer drug failures in clinical trials. Heterotypic multicellular spheroids are three-dimensional (3D) spherical structures generated by self-assembly from aggregates of two or more cell types. Compared to traditional monolayer cell culture models, the organization of cells into a 3D tissue-like structure favors relevant physiological conditions with chemical and physical gradients as well as cell-cell and cell-extracellular matrix (ECM) interactions that recapitulate many of the hallmarks of cancer in situ. Epidermal growth factor receptor (EGFR) mutations are prevalent in non-small cell lung cancer (NSCLC), yet various mechanisms of acquired resistance, including epithelial-to-mesenchymal transition (EMT), limit the clinical benefit of EGFR tyrosine kinase inhibitors (EGFRi). Improved preclinical models that incorporate the complexity induced by epithelial-to-mesenchymal plasticity (EMP) are urgently needed to advance new therapeutics for clinical NSCLC management. This study was designed to provide a thorough characterization of multicellular spheroids of isogenic cancer cells of various phenotypes and demonstrate proof-of-principle for the applicability of the presented spheroid model to evaluate the impact of cancer cell phenotype in drug screening experiments through high-dimensional and spatially resolved imaging mass cytometry (IMC) analyses. First, we developed and characterized 3D homotypic and heterotypic spheroid models comprising EGFRi-sensitive or EGFRi-resistant NSCLC cells. We observed that the degree of EMT correlated with the spheroid generation efficiency in monocultures. In-depth characterization of the multicellular heterotypic spheroids using immunohistochemistry and high-dimensional single-cell analyses by IMC revealed intrinsic differences between epithelial and mesenchymal-like cancer cells with respect to self-sorting, spatiotemporal organization, and stromal cell interactions when co-cultured with fibroblasts. While the carcinoma cells harboring an epithelial phenotype self-organized into a barrier sheet surrounding the fibroblasts, mesenchymal-like carcinoma cells localized to the central hypoxic and collagen-rich areas of the compact heterotypic spheroids. Further, deep-learning-based single-cell segmentation of IMC images and application of dimensionality reduction algorithms allowed a detailed visualization and multiparametric analysis of marker expression across the different cell subsets. We observed a high level of heterogeneity in the expression of EMT markers in both the carcinoma cell populations and the fibroblasts. Our study supports further application of these models in pre-clinical drug testing combined with complementary high-dimensional single-cell analyses, which in turn can advance our understanding of the impact of cancer-stroma interactions and epithelial phenotypic plasticity on innate and acquired therapy resistance in NSCLC.

CAR Co-Operates With Integrins to Promote Lung Cancer Cell Adhesion and Invasion.

The coxsackie and adenovirus receptor (CAR) is a member of the junctional adhesion molecule (JAM) family of adhesion receptors and is localised to epithelial cell tight and adherens junctions. CAR has been shown to be highly expressed in lung cancer where it is proposed to promote tumor growth and regulate epithelial mesenchymal transition (EMT), however the potential role of CAR in lung cancer metastasis remains poorly understood. To better understand the role of this receptor in tumor progression, we manipulated CAR expression in both epithelial-like and mesenchymal-like lung cancer cells. In both cases, CAR overexpression promoted tumor growth in vivo in immunocompetent mice and increased cell adhesion in the lung after intravenous injection without altering the EMT properties of each cell line. Overexpression of WTCAR resulted in increased invasion in 3D models and enhanced β1 integrin activity in both cell lines, and this was dependent on phosphorylation of the CAR cytoplasmic tail. Furthermore, phosphorylation of CAR was enhanced by substrate stiffness in vitro, and CAR expression increased at the boundary of solid tumors in vivo. Moreover, CAR formed a complex with the focal adhesion proteins Src, Focal Adhesion Kinase (FAK) and paxillin and promoted activation of the Guanine Triphosphate (GTP)-ase Ras-related Protein 1 (Rap1), which in turn mediated enhanced integrin activation. Taken together, our data demonstrate that CAR contributes to lung cancer metastasis via promotion of cell-matrix adhesion, providing new insight into co-operation between cell-cell and cell-matrix proteins that regulate different steps of tumorigenesis.

Reversing the Epithelial-Mesenchymal Transition in Metastatic Cancer Cells Using CD146-Targeted Black Phosphorus Nanosheets and a Mild Photothermal Treatment.

Cancer metastasis leads to most deaths in cancer patients, and the epithelial-mesenchymal transition (EMT) is the key mechanism that endows the cancer cells with strong migratory and invasive abilities. Here, we present a nanomaterial-based approach to reverse the EMT in cancer cells by targeting an EMT inducer, CD146, using engineered black phosphorus nanosheets (BPNSs) and a mild photothermal treatment. We demonstrate this approach can convert highly metastatic, mesenchymal-type breast cancer cells to an epithelial phenotype (i.e., reversing EMT), leading to a complete stoppage of cancer cell migration. By using advanced nanomechanical and super-resolution imaging, complemented by immunoblotting, we validate the phenotypic switch in the cancer cells, as evidenced by the altered actin organization and cell morphology, downregulation of mesenchymal protein markers, and upregulation of epithelial protein markers. We also elucidate the molecular mechanism behind the reversal of EMT. Our results reveal that CD146-targeted BPNSs and a mild photothermal treatment synergistically contribute to EMT reversal by downregulating membrane CD146 and perturbing its downstream EMT-related signaling pathways. Considering CD146 overexpression has been confirmed on the surface of a variety of metastatic, mesenchymal-like cancer cells, this approach could be applicable for treating various cancer metastasis via modulating the phenotype switch in cancer cells.

A novel 3D atomistic-continuum cancer invasion model: In silico simulations of an in vitro organotypic invasion assay

We develop a three-dimensional genuinely hybrid atomistic-continuum model that describes the invasive growth dynamics of individual cancer cells in tissue. The framework explicitly accounts for phenotypic variation by distinguishing between cancer cells of an epithelial-like and a mesenchymal-like phenotype. It also describes mutations between these cell phenotypes in the form of epithelial-mesenchymal transition (EMT) and its reverse process mesenchymal-epithelial transition (MET). The proposed model consists of a hybrid system of partial and stochastic differential equations that describe the evolution of epithelial-like and mesenchymal-like cancer cells, respectively, under the consideration of matrix-degrading enzyme concentrations and the extracellular matrix density. With the help of inverse parameter estimation and a sensitivity analysis, this three-dimensional model is then calibrated to an in vitro organotypic invasion assay experiment of oral squamous cell carcinoma cells.

Distinct Ring1b complexes defined by DEAD-box helicases and EMT transcription factors synergistically enhance E-cadherin silencing in breast cancer

Ring1b is a core subunit of polycomb repressive complex 1 (PRC1) and is essential in several high-risk cancers. However, the epigenetic mechanism of Ring1b underlying breast cancer malignancy is poorly understood. In this study, we showed increased expression of Ring1b promoted metastasis by weakening cell–cell adhesions of breast cancer cells. We confirmed that Ring1b could downregulate E-cadherin and contributed to an epigenetic rewiring via PRC1-dependent function by forming distinct complexes with DEAD-box RNA helicases (DDXs) or epithelial-mesenchymal transition transcription factors (EMT TFs) on site-specific loci of E-cadherin promoter. DDXs-Ring1b complexes moderately inhibited E-cadherin, which resulted in an early hybrid EMT state of epithelial cells, and EMT TFs-Ring1b complexes cooperated with DDXs-Ring1b complexes to further repress E-cadherin in mesenchymal-like cancer cells. Clinically, high expression of Ring1b with DDXs or EMT TFs predicted low levels of E-cadherin, metastatic behavior, and poor prognosis. These findings provide an epigenetic regulation mechanism of Ring1b complexes in E-cadherin expression. Ring1b complexes may be potential therapeutic targets and biomarkers for diagnosis and prognosis in invasion breast cancer.

LncRNA LINC00240 suppresses invasion and migration in non-small cell lung cancer by sponging miR-7-5p

BackgroundlncRNAs have important roles in regulating cancer biology. Accumulating evidence has established a link between the dysregulation of lncRNAs and microRNA in cancer progression. In previous studies, miR-7-5p has been found to be significantly down-regulated in mesenchymal-like lung cancer cell lines and directly regulated EGFR. In this work, we investigated the lncRNA partner of miR-7-5p in the progression of lung cancer.MethodsWe investigated the expression of miR-7-5p and the lncRNA after transfection with an miR-7-5p mimics using a microarray. The microarray results were validated using quantitative real time-polymerase Chain Reaction (qRT-PCR). The regulatory effects of lncRNA on miR-7-5p and its target were evaluated by changes in the expression of miR-7-5p after transfection with siRNAs for lncRNA and the synthesis of full-length lncRNA. The effect of miR-7-5p on lncRNA and the miRNA target was evaluated after transfection with miRNA mimic and inhibitor. The role of lncRNA in cancer progression was determined using invasion and migration assays. The level of lncRNA and EGFR in lung cancer and normal lung tissue was analyzed using TCGA data.ResultsWe found that LINC00240 was downregulated in lung cancer cell line after miR-7-5p transfection with an miR-7-5p mimic. Further investigations revealed that the knockdown of LINC00240 induced the overexpression of miR-7-5p. The overexpression of miR-7-5p diminished cancer invasion and migration. The EGFR expression was down regulated after siRNA treatment for LINC00240. Silencing LINC00240 suppressed the invasion and migration of lung cancer cells, whereas LINC00240 overexpression exerted the opposite effect. The lower expression of LINC00240 in squamous lung cancer was analyzed using TCGA data.ConclusionsTaken together, LINC00240 acted as a sponge for miR-7-5p and induced the overexpression of EGFR. LINC00240 may represent a potential target for the treatment of lung cancer.

The ability of miRNAs to induce mesenchymal-to-epithelial transition (MET) in cancer cells is highly dependent upon genetic background.

Understanding of the molecular basis of host cell-miRNA interactions is prerequisite to the successful application of miRNAs as potential therapeutic agents. We studied the morphological and molecular consequences of over expression of three sequence divergent miRNAs previously implicated in the mesenchymal-to-epithelial transition process (MET) in three distinct mesenchymal-like cancer cell lines. The ability of miRNAs to induce morphological changes characteristic of MET positively correlated with induced changes in the expression of genes previously implicated in the process. Variability in the responses of different mesenchymal-like cells to over expression of the same miRNAs was attributable to inherent differences in trans-regulatory profiles pre-disposing these cells to miRNA-induced MET. Collectively our results indicate that miRNA-mediated regulation of MET is a highly integrated process that is significantly modulated by the molecular background of individual cells.

Abstract 759: microRNA 26a-5p inhibited stemness and enhanced the chemosensitivity of non-small cell lung cancer

Abstract Only a subpopulation of cells which have the ability to regulate self-renewal and differentiation of cancer cells, are able to tumorigenic capacity. This subpopulation of cells is called cancer stem cells (CSC). Cancer stem cells are also identified in lung cancer and exhibits resistance to chemotherapy, radiotherapy and targeted therapy. Therefore, the technology to control CSCs is a good strategy to overcome resistance to cancer therapy. Epithelial-mesenchymal transition (EMT) has been linked with the generation of cancer stem cells (CSCs). Recent evidence proposes that EMT-related transcriptomic alterations correlate with the acquisition of CSC phenotype. In previous study, we investigated the miRNA profiles of mesenchymal-like lung cancer cell lines to evaluate the relationship beween EMT and miRNA in non-small cell lung cancer (NSCLC). miR-26a-5p is down-regulated in mesenchymal like lung cancer cell lines. These findings suggested miR-26a-5p may be involved in cancer stemness of lung cancer cells. To test that miR-26a-5p has a role in stemness of lung cancer, we performed colony formation assay, sphere formation assay and fluorescence activated cell sorting. RNA polymerase III subunit G (POLR3G) was selected as a candidate target of miR-26a-5p related to cancer stemness. To verify that miR-26a-5p regulates POLR3G, we performed qRT-PCR, western blot and luciferase assays. Cell proliferation assay was performed to evaluate miR-26a-5p’s role of chemosensitizer. The overexpression of miR-26a-5p induced a marked reduction of the colony formation and sphere formation in non-small lung cancer cell lines. Overexpression of miR-26a-5p lead to reduction of CD133+ cells. Co-treatment with miR-26-5p and Paclitaxel treatment showed a significant reduction of growth. miR-26a-5p regulated the expression of POLR3G at the mRNA and protein levels. These data demonstrate for the first time that miR-26a-5p suppresses lung cancer stemness by repressing POLR3G expression. This provides new insight into a potential approach for the treatment of lung cancer by regulating cancer stemness. Citation Format: Jiwoong Son, Sun Jung Kwon, In Beom Jeong, Ji Hye Kim. microRNA 26a-5p inhibited stemness and enhanced the chemosensitivity of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 759.