Matrix mechanics regulates epithelial defence against cancer by tuning dynamic localization of filamin

In epithelia, normal cells recognize and extrude out newly emerged transformed cells by competition. This process is the most fundamental epithelial defence against cancer, whose occasional failure promotes oncogenesis. However, little is known about what factors determine the success or failure of this defence. Here we report that mechanical stiffening of extracellular matrix attenuates the epithelial defence against HRasV12-transformed cells. Using photoconversion labelling, protein tracking, and loss-of-function mutations, we attribute this attenuation to stiffening-induced perinuclear sequestration of a cytoskeletal protein, filamin. On soft matrix mimicking healthy epithelium, filamin exists as a dynamically single population, which moves to the normal cell-transformed cell interface to initiate the extrusion of transformed cells. However, on stiff matrix mimicking fibrotic epithelium, filamin redistributes into two dynamically distinct populations, including a new perinuclear pool that cannot move to the cell-cell interface. A matrix stiffness-dependent differential between filamin-Cdc42 and filamin-perinuclear cytoskeleton interaction controls this distinctive filamin localization and hence, determines the success or failure of epithelial defence on soft versus stiff matrix. Together, our study reveals how pathological matrix stiffening leads to a failed epithelial defence at the initial stage of oncogenesis.

GRWD1-WDR5-MLL2 Epigenetic Complex Mediates H3K4me3 Mark and Is Essential for Kaposi’s Sarcoma-Associated Herpesvirus-Induced Cellular Transformation

By performing a genome-wide CRISPR-Cas9 screening, we have identified cellular epigenetic regulators that are essential for KSHV-induced cellular transformation. Among them, GRWD1 regulates epigenetic active mark H3K4me3 by interacting with WDR5 and MLL2 and recruiting them to chromatin loci of specific genes in KSHV-transformed cells.

Alterations of Ion Homeostasis in Cancer Metastasis: Implications for Treatment

We have previously reported that metastases from all malignancies are characterized by a core program of gene expression that suppresses extracellular matrix interactions, induces vascularization/tissue remodeling, activates the oxidative metabolism, and alters ion homeostasis. Among these features, the least elucidated component is ion homeostasis. Here we review the literature with the goal to infer a better mechanistic understanding of the progression-associated ionic alterations and identify the most promising drugs for treatment. Cancer metastasis is accompanied by skewing in calcium, zinc, copper, potassium, sodium and chloride homeostasis. Membrane potential changes and water uptake through Aquaporins may also play roles. Drug candidates to reverse these alterations are at various stages of testing, with some having entered clinical trials. Challenges to their utilization comprise differences among tumor types and the involvement of multiple ions in each case. Further, adverse effects may become a concern, as channel blockers, chelators, or supplemented ions will affect healthy and transformed cells alike.

Dissecting RAS Oncogene-Induced Kinome Involved in Breast Cancer Metastasis

Background and Hypothesis: The RAS and PI3K-AKT-mTOR signaling pathways are often dysregulated in cancer. RAS pathway alterations, however, are more common in breast cancer metastasis. The laboratory’s recently developed model system demonstrated the ability of RAS but not PIK3CA-induced signals in promoting metastasis of breast cancer. Unbiased kinome analyses of isogenic RAS-transformed primary tumor and metastatic cells and PIK3CA-transformed primary tumor cells enabled identification of RAS-activated kinome, which included FER, PAK4, LIMK1, PIK3CD and Casein Kinase 2 (CK2). We hypothesized that therapeutic targeting of these kinases may reduce breast cancer metastasis. As a proof-of-principle, the effect of the CK2 inhibitor Silmitasertib, which is in clinical trial for COVID-19 and refractory multiple myeloma, was tested. Experimental Design: The study included four isogenic cell lines: “normal” (KTB34-hTERT), PIK3CA-transformed (TKTB34-PIK3CA), RAS-transformed (TKTB34-RAS), and RAS-transformed cells metastasized to lungs (MKTB34-RAS). Active kinomes in these cells were identified using phospho-proteomics and functional kinome profiling using multiplexed kinase inhibitor beads. Expression levels of FER, PAK4, LIMK1, and PIK3CD kinases were compared through Western Blot using the phospho-antibodies as an indicator of kinase activation. Sensitivity to Silmitasertib was measured using the BrdU Cell Proliferation Assay. Results: FER, PAK4, LIMK1, and PIK3CD were all overexpressed in the TKTB34-RAS and MKTB34-RAS cells compared to KTB34-hTERT and TKTB34-PIK3CA cells. The tested concentration range for Silmitasertib (500 nM to 5 µM) was ineffective in killing the RAS-transformed cells and was overly toxic to “normal” cells. Conclusion and Potential Impact: FER, PAK4, LIMK1, PIK3CD, and CK2 are potential therapeutic targets for breast cancer metastasis. However, Silmitasertib may not be a good candidate as it is more toxic to “normal” cells compared to cancer cells. The isogenic “normal” and transformed cell line model system described here may help to discover new targets and drugs that kill cancer but not normal cells.

Old and New Players of Inflammation and Their Relationship With Cancer Development

Pathogens or genotoxic agents continuously affect the human body. Acute inflammatory reaction induced by a non-sterile or sterile environment is triggered for the efficient elimination of insults that caused the damage. According to the insult, pathogen-associated molecular patterns, damage-associated molecular patterns, and homeostasis-altering molecular processes are released to facilitate the arrival of tissue resident and circulating cells to the injured zone to promote harmful agent elimination and tissue regeneration. However, when inflammation is maintained, a chronic phenomenon is induced, in which phagocytic cells release toxic molecules damaging the harmful agent and the surrounding healthy tissues, thereby inducing DNA lesions. In this regard, chronic inflammation has been recognized as a risk factor of cancer development by increasing the genomic instability of transformed cells and by creating an environment containing proliferation signals. Based on the cancer immunoediting concept, a rigorous and regulated inflammation process triggers participation of innate and adaptive immune responses for efficient elimination of transformed cells. When immune response does not eliminate all transformed cells, an equilibrium phase is induced. Therefore, excessive inflammation amplifies local damage caused by the continuous arrival of inflammatory/immune cells. To regulate the overstimulation of inflammatory/immune cells, a network of mechanisms that inhibit or block the cell overactivity must be activated. Transformed cells may take advantage of this process to proliferate and gradually grow until they become preponderant over the immune cells, preserving, increasing, or creating a microenvironment to evade the host immune response. In this microenvironment, tumor cells resist the attack of the effector immune cells or instruct them to sustain tumor growth and development until its clinical consequences. With tumor development, evolving, complex, and overlapping microenvironments are arising. Therefore, a deeper knowledge of cytokine, immune, and tumor cell interactions and their role in the intricated process will impact the combination of current or forthcoming therapies.

Sustained AMPK Activation and Proline Metabolism Play Critical Roles in the Survival of Matrix-Deprived Transformed Cells

Attachment to the matrix is critical for the survival of adherent cells, whereas detachment triggers death by apoptosis. Therefore, solid tumors must acquire the ability to survive the stress of matrix-detachment to transit through circulation and seed metastases. Although a central role for energy metabolism in cancer progression is well established, what distinguishes its role in the cellular state of the matrix-deprived form compared to the matrix-attached form is not fully understood yet. Using an in vitro transformation model dependent on simian virus 40 (SV40) small t (ST) antigen for cellular survival and proliferation in matrix-deprived conditions, we demonstrate that 5′-adenosine monophosphate-activated protein kinase (AMPK) activity is elevated and sustained under matrix-deprived conditions in ST-expressing fibroblasts. Additionally, these cells display elevated energy (ATP) levels under matrix-deprived conditions in contrast to cells lacking ST expression. The elevated ATP levels are coupled to increased levels of proline in ST-expressing cells, as revealed by metabolomics studies. The AMPK-dependent upregulation of proline oxidase, an enzyme of proline degradation, is a key link for elevated ATP levels. This functional link is further established by proline supplementation concomitant with AMPK activation in matrix-deprived cells lacking ST antigen, yielding ATP and enhancing survival. Thus, our data establishes a key role for AMPK-dependent regulation of proline metabolism in mediating energy homeostasis and promoting survival of matrix-deprived cells. These findings identify key markers that distinguish the metabolic states of matrix-detached and matrix-attached transformed cells and have implications in developing novel therapeutic strategies for specifically targeting matrix-detached metastasizing cancer cells.

The roles of distinct Ca2+ signaling mediated by Piezo and inositol triphosphate receptor (IP3R) in the remodeling of E-cadherin during cell dissemination

Given the role of E-cadherin (E-cad) in holding epithelial cells together, the inverse relationship between E-cad levels and cell invasion has been perceived as a principle underlying the invasiveness of tumor cells. In contrast, our study employing the Drosophila model of cell dissemination demonstrates that E-cad is necessary for the invasiveness of RasV12-transformed cells in vivo. Drosophila E-cad/β-catenin disassembles at adherens junctions and assembles at invasive protrusions—the actin- and cortactin-rich invadopodia-like protrusions associated with breach of the extracellular matrix (ECM)—during cell dissemination. Loss of E-cad attenuates dissemination of RasV12-transformed cells by impairing their ability to compromise the ECM. Strikingly, the remodeling of E-cad/β-catenin subcellular distribution is controlled by two discrete intracellular calcium signaling pathways: Ca2+ release from endoplasmic reticulum via the inositol triphosphate receptor (IP3R) disassembles E-cad at adherens junctions while Ca2+ entry via the mechanosensitive channel Piezo assembles E-cad at invasive protrusions. Thus, our study provides molecular insights into the unconventional role of E-cad in cell invasion during cell dissemination in vivo and describes the discrete roles of intracellular calcium signaling in the remodeling of E-cad/β-catenin subcellular localization.

PI3K/AKT Activation Mediates B-Cell Transformation By the "T" Splice-Variant of Fyn Kinase

The SRC-family kinase FYN has key roles downstream of growth-factor receptor activation and is implicated in various cancers. Activating FYNutations were reported in angioimmunoblastic T-cell lymphoma (AITL), but its role in transforming lymphocytes is poorly characterized. FYN also may play a role in signaling downstream of the B cell Receptor (BCR) receptor in Diffuse Large B-Cell Lymphoma (DLBCL), and laboratory studies show inhibition of FYN and other SRC family kinases (SFKs) can block BCR-mediated lymphomagenesis. To investigate FYN's role in oncogenic singling, we introduced FYN alleles to IL3-dependent FL5.12 murine pro-B cells and tested for selective advantage upon cytokine withdrawal and ability to transform the cells to cytokine independence. FYN B and T variants result from alternate splicing of exon 7, with B originally detected in brain and T in hematopoietic cells, but both are expressed and may be deregulated associated with disease states in diffuse tissues. We employed wildtype (WT) alleles for both spliceoforms along with mutations detected recurrently in AITL (L174R and R176C), kinase active (Y528H), and kinase dead (K299A). No FYN B alleles showed any selective advantage in the prolymphocyte cells subjected to cytokine withdrawal. In contrast, all FYN T alleles other than kinase dead were strongly enriched through multiple rounds of IL3 withdrawal and rescue. Indeed, complete withdrawal of cytokine resulted in transformation to IL3-independent growth, generating stable cell lines addicted to FYN kinase activity for survival. The SFK inhibitor PP2 was toxic to all FYN-T transformed cells but not to baseline FL5.12 cells growing in cytokine. WT- and L147R-transformed cells were similarly sensitive to PP2, while the other AITL-derived mutant R176C demonstrated reduced sensitivity. Immunoblotting demonstrated PP2-sensitive phosphorylation of AKT and the MTORC1 target 4EBP1, suggesting PI3K/AKT activation as a key survival pathway downstream from transformation by FYN T alleles. Consistently, the AKT inhibitor ipatasertib and PDK1 inhibitor GSK2334470 were highly active against all FYN-transformed lines, and AKT phosphorylation was sensitive to PDK1 inhibition, showing convergence on AKT activation in all transformants. Pan-PI3K inhibition was active against FYN transformants, while the delta isoform-specific inhibitor idelalisib showed only partial activity, suggesting activation of multiple PI3K isoforms downstream of FYN activity. These results suggest that AKT, PI3K, and PDK1 are important targets downstream of FYN, and that the PI3K/AKT pathway is the key survival mechanism in lymphomagenesis mediated by FYN. We show that WT and activating mutants of the FYN T spliceoform specifically, but not FYN B, induce drug-sensitive B lymphocyte malignant transformation by activating PI3K/AKT/MTORC1 signaling. Disclosures No relevant conflicts of interest to declare.

Human myeloma RPMI8226 cells and normal cells have different sensitivity to bisilicate silver nanoparticles in vitro

Introduction. Silver nanoparticles due to its pronounced cytotoxicity are regarded as promising agent for anticancer therapy. Determination of normal and transformed cells sensitivity to silver nanoparticles can be the basis for the application as an adjuvant cancer treatment. The objective of the study was to investigate influence of atomic clusters of Argentum (ACA) in the form of silver bisilicate nanoparticles colloid solution on viability and proliferation of human myeloma cell line, mesenchymal stromal cells and blood lymphocytes. Material and methods. Cell viability was evaluated by MTT and LDH assay. Cell proliferation was evaluated by flow cytometry. Results. It was found that ACA had dose-depending cytotoxicity toward all investigated cell types, but normal and transformed cells varied significantly in the sensitivity to nanoparticles. IC50 for myeloma cell line RPMI8226 was 1,75 µg/ml. For MSCs of different origin IC50 was in the range of 12 to 16 µg/ml. ACA in concentration from 2 to 3 µg/ml induced RPMI8226 cells metabolic disruption and death without influence on viability and cell cycle of mesenchymal stromal cells and blood lymphocytes. Conclusion. Results of work has shown distinct differences in sensitivity to ACA between myeloma cells, mesenchymal stromal cells and blood lymphocytes. The optimal range of ACA concentration with anticancer effect without cytotoxic influence on normal cells has been determined in vitro.