Oncogenic microRNA-1290 and SCAI Gene as Potential Biomarker for Colorectal Carcinoma.

INTRODUCTION: Colorectal cancer (CRC) is the world's third most frequent cancer, with a significant mortality rate due to late detection. There is a need to search for biomarkers that can detect colorectal cancer at an early stage. MicroRNAs (miRNAs) regulate several targets that function as oncogenes and/or tumor suppressor genes, so any change in microRNA expression level can predict abnormality. OBJECTIVE: The objective of the study was to evaluate the expression of miR-1290, and Suppressor of cancer cell invasion (SCAI) gene that may be used as biomarkers for early diagnosis of colorectal carcinoma. METHODOLOGY: This study included 50 subjects consisting of newly diagnosed colorectal carcinoma patients (n = 25), and healthy controls (n = 25). After RNA isolation and reverse transcription, the expression level of miR-1290 and SCAI gene in the tissues and plasma samples of CRC patients were analyzed using real time PCR and compared with healthy individuals as normal controls. The 2-ΔΔCt formula was used to compute the fold-change, while using miR-16 and GAPDH as reference genes for normalization. RESULTS: We found that miR-1290 is upregulated, whereas SCAI gene is downregulated in both plasma and tissue samples of CRC patients. For miR-1290, the sensitivity was 96% and specificity was 100%, and for SCAI, 100% sensitivity and 88% specificity was calculated by ROC analysis. CONCLUSION: The expression of miR-1290 and SCAI gene may be utilized as biomarkers for diagnosis of colorectal carcinoma.

Physical network regimes of 3D fibrillar collagen networks trigger invasive phenotypes of breast cancer cells.

The mechanical characteristics of the extracellular environment are known to significantly influence cancer cell behavior in vivo and in vitro. The structural complexity and viscoelastic dynamics of the extracellular matrix (ECM) pose significant challenges in understanding its impact on cancer cells. Herein, we report distinct regulatory signatures in the invasion of different breast cancer cell lines into three-dimensional (3D) fibrillar collagen networks, caused by systematic modifications of the physical network properties. By reconstituting collagen networks of thin fibrils, we demonstrate that such networks can display network strand flexibility akin to that of synthetic polymer networks, known to exhibit entropic rubber elasticity. This finding contrasts with the predominant description of the mechanics of fibrillar collagen networks by an enthalpic bending elasticity of rod-like fibrils. Mean-squared displacement analysis of free-standing fibrils confirmed a flexible fiber regime in networks of thin fibrils. Furthermore, collagen fibrils in both networks were softened by the adsorption of highly negatively charged sulfonated polymers and colloidal probe force measurements of network elastic modulus again proofed the occurrence of the two different physical network regimes. Our cell assays revealed that the cellular behavior (morphology, clustering, invasiveness, matrix metalloproteinase (MMP) activity) of the 'weakly invasive' MCF-7 and 'highly invasive' MDA-MB-231 breast cancer cell lines is distinctively affected by the physical (enthalpic/entropic) network regime, and cannot be explained by changes of the network elastic modulus, alone. These results highlight an essential pathway, albeit frequently overlooked, how the physical characteristics of fibrillar ECMs affect cellular behavior. Considering the coexistence of diverse physical network regimes of the ECM in vivo, our findings underscore their critical role of ECM's physical network regimes in tumor progression and other cell functions, and moreover emphasize the significance of 3D in vitro collagen network models for quantifying cell responses in both healthy and pathological states.

Mechanobiological cell adaptations to changing microenvironments determine cancer invasiveness: Experimentally validated finite element modeling.

Metastases are the leading cause of cancer-associated deaths. A key process in metastasis is cell invasiveness, which is driven and controlled by cancer cell interactions with their microenvironment. We have previously shown that invasive cancer cells forcefully push into and indent physiological stiffness gels to cell-scale depths, where the percentage of indenting cells and their attained depths provide clinically relevant predictions of tumor invasiveness and the potential metastatic risk. The cell-attained, invasive indentation depths are directly affected by gel-microenvironment mechanics, which can concurrently modulate the cells' mechanics and force application capacity, in a complex, coordinated mechanobiological response. As it is impossible to experimentally isolate the different contributions of cell and gel mechanics to cancer cell invasiveness, we perform finite element modeling with literature-based parameters. Under average-scale, cell cytoplasm and nucleus mechanics and cell-applied force levels, increasing gel stiffness 1-50 kPa significantly reduced the attained indentation depth by >200%, while the gel's Poisson ratio reduced depths only by up to 20% and only when the ratio was >0.4; this reveals microenvironment mechanics that can promote invasiveness. Experiments with varying-invasiveness cancer cells exhibited qualitative variations in their responses to gel stiffness increase, for example large/small reduction in indentation depth or increase and then reduction. We quantitatively and qualitatively reproduced the different experimental responses via coordinated changes in cell mechanics and applied force levels. Thus, the different cancer cell capacities to adapt their mechanobiology in response to mechanically changing microenvironments likely determine the varying cancer invasiveness and metastatic risk levels in patients.

Role of Calcium-Activated Potassium Channels in Proliferation, Migration and Invasion of Human Chronic Myeloid Leukemia K562 Cells.

Calcium-activated potassium channels (KCa) are important participants in calcium signaling pathways due to their ability to be activated by an increase in intracellular free calcium concentration. KCa channels are involved in the regulation of cellular processes in both normal and pathophysiological conditions, including oncotransformation. Previously, using patch-clamp, we registered the KCa currents in the plasma membrane of human chronic myeloid leukemia K562 cells, whose activity was controlled by local Ca2+ entry via mechanosensitive calcium-permeable channels. Here, we performed the molecular and functional identification of KCa channels and have uncovered their role in the proliferation, migration and invasion of K562 cells. Using a combined approach, we identified the functional activity of SK2, SK3 and IK channels in the plasma membrane of the cells. Selective SK and IK channel inhibitors, apamin and TRAM-34, respectively, reduced the proliferative, migratory and invasive capabilities of human myeloid leukemia cells. At the same time, the viability of K562 cells was not affected by KCa channel inhibitors. Ca2+ imaging showed that both SK and IK channel inhibitors affect Ca2+ entry and this could underlie the observed suppression of pathophysiological reactions of K562 cells. Our data imply that SK/IK channel inhibitors could be used to slow down the proliferation and spreading of chronic myeloid leukemia K562 cells that express functionally active KCa channels in the plasma membrane.

Syndecan-4 is a maestro of gastric cancer cell invasion and communication that underscores poor survival.

Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options. Here, we show that syndecan-4 (SDC4), a transmembrane proteoglycan, is highly expressed in intestinal subtype gastric tumors and that this signature associates with patient poor survival. Further, we mechanistically demonstrate that SDC4 is a master regulator of gastric cancer cell motility and invasion. We also find that SDC4 decorated with heparan sulfate is efficiently sorted in extracellular vesicles (EVs). Interestingly, SDC4 in EVs regulates gastric cancer cell-derived EV organ distribution, uptake, and functional effects in recipient cells. Specifically, we show that SDC4 knockout disrupts the tropism of EVs for the common gastric cancer metastatic sites. Our findings set the basis for the molecular implications of SDC4 expression in gastric cancer cells and provide broader perspectives on the development of therapeutic strategies targeting the glycan-EV axis to limit tumor progression.

Drug Resistance in Medulloblastoma Is Driven by YB-1, ABCB1 and a Seven-Gene Drug Signature.

Therapy resistance represents an unmet challenge in the treatment of medulloblastoma. Accordingly, the identification of targets that mark drug-resistant cell populations, or drive the proliferation of resistant cells, may improve treatment strategies. To address this, we undertook a targeted approach focused on the multi-functional transcription factor YB-1. Genetic knockdown of YB-1 in Group 3 medulloblastoma cell lines diminished cell invasion in 3D in vitro assays and increased sensitivity to standard-of-care chemotherapeutic vincristine and anti-cancer agents panobinostat and JQ1. For vincristine, this occurred in part by YB-1-mediated transcriptional regulation of multi-drug resistance gene ABCB1, as determined by chromatin immunoprecipitation. Whole transcriptome sequencing of YB-1 knockdown cells identified a role for YB-1 in the regulation of tumourigenic processes, including lipid metabolism, cell death and survival and MYC and mTOR pathways. Stable cisplatin- and vincristine-tolerant Group 3 and SHH cell lines were generated to identify additional mechanisms driving resistance to standard-of-care medulloblastoma therapy. Next-generation sequencing revealed a vastly different transcriptomic landscape following chronic drug exposure, including a drug-tolerant seven-gene expression signature, common to all sequenced drug-tolerant cell lines, representing therapeutically targetable genes implicated in the acquisition of drug tolerance. Our findings provide significant insight into mechanisms and genes underlying therapy resistance in medulloblastoma.

Sequential genome-wide CRISPR-Cas9 screens identify genes regulating cell-surface expression of tetraspanins.

Tetraspanins, a superfamily of membrane proteins, mediate diverse biological processes through tetraspanin-enriched microdomains in the plasma membrane. However, how their cell-surface presentation is controlled remains unclear. To identify the regulators of tetraspanin trafficking, we conduct sequential genome-wide loss-of-function CRISPR-Cas9 screens based on cell-surface expression of a tetraspanin member, TSPAN8. Several genes potentially involved in endoplasmic reticulum (ER) targeting, different biological processes in the Golgi apparatus, and protein trafficking are identified and functionally validated. Importantly, we find that biantennary N-glycans generated by MGAT1/2, but not more complex glycan structures, are important for cell-surface tetraspanin expression. Moreover, we unravel that SPPL3, a Golgi intramembrane-cleaving protease reported previously to act as a sheddase of multiple glycan-modifying enzymes, controls cell-surface tetraspanin expression through a mechanism associated with lacto-series glycolipid biosynthesis. Our study provides critical insights into the molecular regulation of cell-surface presentation of tetraspanins with implications for strategies to manipulate their functions, including cancer cell invasion.

Multimodal Techniques to Study Tumor Growth, Basement Membrane Breaching, and Invasion in 3D Matrices.

Cancer-derived organoids and three-dimensional (3D) extracellular matrix (ECM) are taking center stage as in vitro models to study neoplastic cell behavior, since they recapitulate the heterogeneous cellular composition of tumors and their extracellular environment. In combination with imaging and molecular/biochemical techniques, 3D organoid models have contributed substantially to our knowledge about the cellular and molecular mechanisms that regulate the growth of tumors and invasion into the surrounding tissue. We here outline a set of protocols that describe culturing of cancer-derived organoids in 3D matrices and various strategies that allow modeling of tumor growth, tumor cell penetration into basement membranes, and invasion into Collagen I-rich ECM. Furthermore, we specify protocols for subsequent handling of organoids cultured in 3D ECM for confocal microscopy and analysis of gene expression at the protein and mRNA level. Although we here use breast cancer-derived organoids, these protocols can be directly applied or adapted for organoids derived from other cancer types or healthy tissues. Thus, in addition to investigating cell behavior of multiple cancer types, the combination of protocols described here may be used to study processes such as cell differentiation and migration during homeostasis and normal development.

Structural Insights Uncover the Specific Phosphoinositide Recognition by the PH1 Domain of Arap3.

Arap3, a dual GTPase-activating protein (GAP) for the small GTPases Arf6 and RhoA, plays key roles in regulating a wide range of biological processes, including cancer cell invasion and metastasis. It is known that Arap3 is a PI3K effector that can bind directly to PI(3,4,5)P3, and the PI(3,4,5)P3-mediated plasma membrane recruitment is crucial for its function. However, the molecular mechanism of how the protein recognizes PI(3,4,5)P3 remains unclear. Here, using liposome pull-down and surface plasmon resonance (SPR) analysis, we found that the N-terminal first pleckstrin homology (PH) domain (Arap3-PH1) can interact with PI(3,4,5)P3 and, with lower affinity, with PI(4,5)P2. To understand how Arap3-PH1 and phosphoinositide (PIP) lipids interact, we solved the crystal structure of the Arap3-PH1 in the apo form and complex with diC4-PI(3,4,5)P3. We also characterized the interactions of Arap3-PH1 with diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 in solution by nuclear magnetic resonance (NMR) spectroscopy. Furthermore, we found overexpression of Arap3 could inhibit breast cancer cell invasion in vitro, and the PIPs-binding ability of the PH1 domain is essential for this function.

Radiosensitization with Gadolinium Chelate-Coated Gold Nanoparticles Prevents Aggressiveness and Invasiveness in Glioblastoma.

Purpose: This study aimed to evaluate the radiosensitizing potential of Au@DTDTPA(Gd) nanoparticles when combined with conventional external X-ray irradiation (RT) to treat GBM. Methods: Complementary biological models based on U87 spheroids including conventional 3D invasion assay, organotypic brain slice cultures, chronic cranial window model were implemented to investigate the impact of RT treatments (10 Gy single dose; 5×2 Gy or 2×5 Gy) combined with Au@DTDTPA(Gd) nanoparticles on tumor progression. The main tumor mass and its infiltrative area were analyzed. This work focused on the invading cancer cells after irradiation and their viability, aggressiveness, and recurrence potential were assessed using mitotic catastrophe quantification, MMP secretion analysis and neurosphere assays, respectively. Results: In vitro clonogenic assays showed that Au@DTDTPA(Gd) nanoparticles exerted a radiosensitizing effect on U87 cells, and in vivo experiments suggested a benefit of the combined treatment "RT 2×5 Gy + Au@DTDTPA(Gd)" compared to RT alone. Invasion assays revealed that invasion distance tended to increase after irradiation alone, while the combined treatments were able to significantly reduce tumor invasion. Monitoring of U87-GFP tumor progression using organotypic cultures or intracerebral grafts confirmed the anti-invasive effect of Au@DTDTPA(Gd) on irradiated spheroids. Most importantly, the combination of Au@DTDTPA(Gd) with irradiation drastically reduced the number, the viability and the aggressiveness of tumor cells able to escape from U87 spheroids. Notably, the combined treatments significantly reduced the proportion of escaped cells with stem-like features that could cause recurrence. Conclusion: Combining Au@DTDTPA(Gd) nanoparticles and X-ray radiotherapy appears as an attractive therapeutic strategy to decrease number, viability and aggressiveness of tumor cells that escape and can invade the surrounding brain parenchyma. Hence, Au@DTDTPA(Gd)-enhanced radiotherapy opens up interesting perspectives for glioblastoma treatment.

Computational modeling reveals a vital role for proximity-driven additive and synergistic cell-cell interactions in increasing cancer invasiveness.

Solid-tumor cell invasion typically occurs by collective migration of attached cell-cohorts, yet we show here that indirect cell-interactions through the substrate can also drive invasiveness. We have previously shown that well-spaced, invasive cancer cells push-into and indent gels to depths of 10 µm, while closely adjacent, non-contacting cancer cells may reach up to 18 µm, potentially relying on cell-cell interactions through the gel-substrate. To test that, we developed finite element models of indenting cells, using experimental gel mechanics, cell mechanostructure, and force magnitudes. We show that under 50-350 nN of combined traction and normal forces, a stiff nucleus-region is essential in facilitating 5-10 µm single-cell indentations, while uniformly soft cells attain 1.6-fold smaller indentations. We observe that indentation depths of cells in close proximity (0.5-50 µm distance) increase relative to well-spaced cells, due to additive, continuum mechanics-driven contributions. Specifically, 2-3 cells applying 220 nN normal forces gained up to 3% in depth, which interestingly increased to 7.8% when two cells, 10 µm apart, applied unequal force-magnitudes (i.e., 220 and 350 nN). Such additive, energy-free contributions can reduce cell mechanical energy -output required for invasiveness, yet the experimentally observed 10-18 µm depths likely necessitate synergistic, mechanobiological changes, which may be mechanically triggered. We note that nucleus stiffening or cytoplasm softening by 25-50% increased indentation depths by only 1-7%, while depths increase nearly linearly with force-magnitude even to two-fold levels. Hence, cell-proximity triggered, synergistic and additive cell-interactions through the substrate can drive collective cancer-cell invasiveness, even without direct cell-cell interactions. STATEMENT OF SIGNIFICANCE: Metastatic cancer invasion typically occurs collectively in attached cell-cohorts. We have previously shown increased invasiveness in closely adjacent cancer cells that are able to push-into and indent soft-gels more deeply than single, well-spaced cells. Using finite element models, we reveal mechanisms of cell-proximity driven invasiveness, demonstrating an important role for the stiff nucleus. Cell-proximity can additively induce small increase in indentation depth via continuum mechanics contributions, especially when adjacent cells apply unequal forces, and without requiring increased cell-mechanical-energy-output. Concurrently, proximity-triggered synergistic interactions that produce changes in cell mechanics or capacity for increased force-levels can facilitate deep invasive-indentations. Thus, we reveal concurrent additive and synergistic mechanisms to drive collective cancer-cell invasiveness even without direct cell-cell interactions.

Glycosyltransferases EXTL2 and EXTL3 cellular balance dictates heparan sulfate biosynthesis and shapes gastric cancer cell motility and invasion.

Heparan sulfate (HS) proteoglycans (HSPGs) are abundant glycoconjugates in cells' glycocalyx and extracellular matrix. By acting as scaffolds for protein-protein interactions, HSPGs modulate extracellular ligand gradients, cell signaling networks, and cell-extracellular matrix crosstalk. Aberrant expression of HSPGs and enzymes involved in HSPG biosynthesis and processing has been reported in tumors, with impact in cancer cell behavior and tumor microenvironment properties. However, the roles of specific glycosyltransferases in the deregulated biosynthesis of HSPGs are not fully understood. In this study, we established glycoengineered gastric cancer cell models lacking either exostosin-like glycosyltransferase 2 (EXTL2) or EXTL3 and revealed their regulatory roles in both HS and chondroitin sulfate (CS) biosynthesis and structural features. We showed that EXTL3 is key for initiating the synthesis of HS chains in detriment of CS biosynthesis, intervening in the fine-tuned balance of the HS/CS ratio in cells, while EXTL2 functions as a negative regulator of HS biosynthesis, with impact over the glycoproteome of gastric cancer cells. We demonstrated that KO of EXTL2 enhanced HS levels along with concomitant upregulation of Syndecan-4, which is a major cell surface carrier of HS. This aberrant HS expression profile promoted a more aggressive phenotype, characterized by higher cellular motility and invasion, and impaired activation of Ephrin type-A 4 cell surface receptor tyrosine kinase. Our findings uncover the biosynthetic roles of EXTL2 and EXTL3 in the regulation of cancer cell GAGosylation and proteoglycans expression and unravel the functional consequences of aberrant HS/CS balance in cellular malignant features.

HIV-1 Proteins gp120 and Tat Promote Epithelial-Mesenchymal Transition and Invasiveness of HPV-Positive and HPV-Negative Neoplastic Genital and Oral Epithelial Cells.

The incidence of human papillomavirus (HPV)-associated anogenital and oropharyngeal cancer in human immunodeficiency virus (HIV)-infected individuals is substantially higher than in HIV-uninfected individuals. HIV may also be a risk factor for the development of HPV-negative head and neck, liver, lung, and kidney cancer. However, the molecular mechanisms underlying HIV-1-associated increase of epithelial malignancies are not fully understood. Here, we showed that HPV-16-immortalized anal AKC-2 and cervical CaSki epithelial cells that undergo prolonged exposure to cell-free HIV-1 virions or HIV-1 viral proteins gp120 and tat respond with the epithelial-mesenchymal transition (EMT) and increased invasiveness. Similar responses were observed in HPV-16-infected SCC-47 and HPV-16-negative HSC-3 oral epithelial cancer cells that were cultured with these viral proteins. EMT induced by gp120 and tat led to detachment of poorly adherent cells from the culture substratum; these cells remained capable of reattachment, upon which they coexpressed both E-cadherin and vimentin, indicative of an intermediate stage of EMT. The reattached cells also expressed stem cell markers CD133 and CD44, which may play a critical role in cancer cell invasion and metastasis. Inhibition of transforming growth factor (TGF)-ß1 and MAPK signaling and vimentin expression, and restoration of E-cadherin expression reduced HIV-induced EMT and the invasive activity of HPV-16-immortalized anal and cervical epithelial cells. Collectively, our results suggest that these approaches along with HIV viral suppression with antiretroviral therapy (ART) might be useful to limit the role of HIV-1 infection in the acceleration of HPV-associated or HPV-independent epithelial neoplasia. IMPORTANCE HPV-16-immortalized genital and oral epithelial cells and HPV-negative oral cancer cells that undergo prolonged contact with cell-free HIV-1 virions or with viral proteins gp120 and tat respond by becoming more invasive. EMT cells induced by HIV-1 in cultures of HPV-16-immortalized anal and cervical epithelial cells express the stem cell markers CD133 and CD44. These results suggest that the interaction of HIV-1 with neoplastic epithelial cells may lead to their de-differentiation into cancer stem cells that are resistant to apoptosis and anti-cancer drugs. Thus, this pathway may play a critical role in the development of invasive cancer. Inhibition of TGF-ß1 and MAPK signaling and vimentin expression, and restoration of E-cadherin expression reduced HIV-induced EMT and the invasiveness of HPV-16-immortalized anal and cervical epithelial cells. Taken together, these results suggest that these approaches might be exploited to limit the role of HIV-1 infection in the acceleration of HPV-associated or HPV-independent epithelial neoplasia.

STAT3 mediates RCP-induced cancer cell invasion through the NF-κB/Slug/MT1-MMP signaling cascade.

Rab coupling protein (RCP) has been known to induce cancer invasion and metastasis, and STAT3 is one of major oncogenic factors. In the present study, we identify the critical role of STAT3 in RCP-induced cancer cell invasion. Immunohistochemical data of ovarian cancer tissues presented that levels of RCP expression are closely correlated with those of phospho-STAT3 (p-STAT3). In addition, ovarian cancer patients with high expression of both RCP and p-STAT3 had significantly lower progress-free and overall survival rates compared to those with low either RCP or p-STAT3 expression. Mechanistically, RCP induced STAT3 phosphorylation in both ovarian and breast cancer cells. Silencing or pharmacological inhibition of STAT3 significantly inhibited RCP-induced cancer cell invasion. In addition, we provide evidence that the ß1 integrin/EGFR axis is important for RCP-induced STAT3 phosphorylation. Furthermore, STAT3 activated NF-κB for Slug expression that in turn upregulated MT1-MMP expression for cancer cell invasion. Collectively, our present data demonstrate that STAT3 is located downstream of the ß1 integrin/EGFR axis and induces Slug and MT1-MMP expression for cancer cell invasion.

A Programmable Multifunctional 3D Cancer Cell Invasion Micro Platform.

In the research of cancer cell invasion and metastasis, recreation of physiologically relevant and faithful three-dimensional (3D) tumor models that recapitulate spatial architecture, spatiotemporal control of cell communication and signaling pathways, and integration of extracellular cues remains an open challenge. Here, a programmable multifunctional 3D cancer cell invasion microbuckets-hydrogel (Mb-H) platform is developed by integrating various function-variable microbuckets and extracellular matrix (ECM)-like hydrogels. Based on this Mb-H micro platform, the aggregation of multi-cancer cells is well controlled to form cancer cell spheroids, and the guiding relationship of single-cell migration and collective cell migration during the epithelial-mesenchymal transition (EMT) of cancer cell invasion are demonstrated. By programming and precisely assembling multiple functions in one system, the Mb-H platform with spatial-temporal controlled release of cytokine transforming growth factor beta (TGF-ß) and various functionalized Mb-H platforms with intelligent adjustment of cell-matrix interactions are engineered to coordinate the 3D invasive migration of cancer cell spheroids. This programmable and adaptable 3D cancer cell invasion micro platform takes a new step toward mimicking the dynamically changing (localized) tumor microenvironment and exhibits wide potential applications in cancer research, bio-fabrication, cell signaling, and drug screening.

A multi-layered hybrid model for cancer cell invasion.

In this article, a hybrid model is developed based on multi-scale concept for solid  tumour cell invasion into a healthy tissue. Our aim is to study the tumour heterogeneity due to the geometry of a growing tumour caused by the phenotypic transformations of cells. In this context, an early vascular growth is considered after angiogenesis. Hence, the microenvironment of the solid tumour is rich of oxygen and nutrients. It is also considered that epidermal growth factor (EGF) is distributed into the surrounding extracellular matrix (ECM) of the tumour. The developed multi-layered model consists of three layers: intracellular or subcellular, cellular, and extracellular or tissue layer. The model integrates the events that occur simultaneously in these three layers to identify the underlying diversity. Here, every cell is represented as an agent. Characteristics of an agent are controlled by its intracellular protein expressions and its surrounding microenvironment. A mature proliferative or migratory or hybrid cell agent spawn two indistinguishable children unless it may convert into other phenotype due to influence of the microenvironment. Further, a simple cell cycle model is adapted which is influenced by EGF-EGFR signalling pathway and the external oxygen and nutrients. Moreover, migratory and hybrid cells secrete several matrix degrading enzymes (MDEs) which remodel the ECM for tumour invasion locally. Several biomechanical forces are considered that simultaneously act on the cancer cells. The outcome of the model is very similar to the results reported in earlier studies. The model shows the characteristics of cancer invasion that include sustainable proliferation by ignoring growth suppressor signals and reproduction of cancer cells at abnormal proportion, restrict apoptosis, and invade into the surrounding tissue. As the simulation parameters get modified due to different biochemical and biophysical processes, the robustness of the model is determined. It is found that only a number of proliferative cells are moderately sensitive to the parameters and others are less-sensitive.

Inhibin ß-A (INHBA) induces epithelial-mesenchymal transition and accelerates the motility of breast cancer cells by activating the TGF-ß signaling pathway.

Accumulating evidence indicates that INHBA (Inhibin ß-A, a member of the TGF-ß superfamily) functions as an oncogene in cancer progression. However, little is known as to how INHBA regulates the progression and aggressiveness of breast cancer (BC). This study explored the function and underlying mechanism of INHBA in epithelial-mesenchymal transition (EMT) of BC cells. INHBA expression in BC cell lines was measured using RT-qPCR and Western blot. The would-healing and transwell migration assays were used to investigate the effect of INHBA overexpression or silencing on BC cell motility. Moreover, the expression levels of EMT-related genes were quantified after overexpressing or silencing of INHBA. Based on published dataset, INHBA was significantly upregulated in BC tissues compared to the adjacent normal tissues. A higher level of INHBA expression was also correlated with a poor survival in BC patients. In addition, in vitro study showed that INHBA played an indispensable role in promoting BC cell proliferation and invasion. Mechanistically, INHBA induced epithelial-mesenchymal transition (EMT) and accelerated the motility of BC cells by activating TGF-ß-regulated genes. In conclusion, INHBA plays a functional role in supporting EMT phenotype of BC cells, and it may serve as a diagnostic biomarker and a potential therapeutic target for BC treatment.

Preferential interaction of platelets with prostate cancer cells with stem cell markers.

BACKGROUND: Prostate cancer (PCa) may be initiated by CD133+/CD44+ expressing stem cell-like cells (PCSC), which are also thought to drive metastasis. Platelets also contribute to metastasis via tumor cell-induced platelet aggregation (TCIPA), which in part enhances cancer cell invasion. Moreover, activated platelets secrete stromal derived growth factor-1α (SDF-1α) that can mobilize CSCs via the CXCR4 receptor. However, the potential reciprocal interactions between CSCs and platelets have not been investigated. OBJECTIVE: To characterize the mechanisms behind PCSC-platelet interaction. METHODS: Fluorescence Activated Cell Sorting was utilized to separate DU145 and PC3 PCa cells into CD133+/CD44+, CD133+/CD44-, CD44+/CD133-, and CD133-/CD44- subpopulations and to measure their CXCR4 surface expression. PCa subpopulation TCIPA experiments were performed using aggregometry and immunoblot was used to measure prothrombin. Platelet SDF-1α secretion was measured by ELISA. Modified-Boyden chamber assays were used to assess the role of SDF-1α:CXCR4 pathway in platelet-PCSC interactions. RESULTS: DU145 and PC3 expressing both CD133 and CD44 stem cell markers accounted for only small fractions of total cells (DU145: CD133+/CD44+ 3.44 ± 1.45% vs. CD133+/CD44- 1.56 ± 0.45% vs. CD44+/CD133- 68.19 ± 6.25% vs. CD133-/CD44- 20.36 ± 4.51%). However, CD133+ subpopulations induced the greatest amount of aggregation compared to CD44+/CD133- and double-negative DU145, and this aggregation potency of CD133+ PCa cells corresponded with high levels of prothrombin expression. Additionally, CD133+ subpopulations expressed significantly higher level of CXCR4 compared to CD133-/CD44- and CD44+/CD133-. Disruption of SDF-1α:CXCR4 pathway reduced platelet-induced PCSC invasion. CONCLUSIONS: CD133+/CD44+ and CD133+/CD44- PCSCs have highest platelet aggregation potency, which could be attributed to their increased prothrombin expression. Reciprocally, platelet-derived SDF-1α stimulates PCSC invasion.

Optogenetic Control of Myocardin-Related Transcription Factor A Subcellular Localization and Transcriptional Activity Steers Membrane Blebbing and Invasive Cancer Cell Motility.

The myocardin-related transcription factor A (MRTF-A) controls the transcriptional activity of the serum response factor (SRF) in a tightly controlled actin-dependent manner. In turn, MRTF-A is crucial for many actin-dependent processes including adhesion, migration, and contractility and has emerged as a novel target for anti-tumor strategies. MRTF-A rapidly shuttles between cytoplasmic and nuclear compartment via dynamic actin interactions within its N-terminal RPEL domain. Here, optogenetics is used to spatiotemporally control MRTF-A nuclear localization by blue light using the light-oxygen-voltage-sensing domain 2-domain based system LEXY (light-inducible nuclear export system). It is found that light-regulated nuclear export of MRTF-A occurs within 10-20 min. Importantly, MRTF-A-LEXY shuttling is independent of perturbations of actin dynamics. Furthermore, light-regulation of MRTF-A-LEXY is reversible and repeatable for several cycles of illumination and its subcellular localization correlates with SRF transcriptional activity. As a consequence, optogenetic control of MRTF-A subcellular localization determines subsequent cytoskeletal dynamics such as non-apoptotic plasma membrane blebbing as well as invasive tumor-cell migration through 3D collagen matrix. This data demonstrates robust optogenetic regulation of MRTF as a powerful tool to control SRF-dependent transcription as well as cell motile behavior.

In situ continuously monitoring of cancer cell invasion process based on impedance sensing.

Activation of invasion and metastasis is recognized as one of the hallmarks of cancer. There are 90% of cancer-related deaths due to metastasis and given that it is worthy of note to study cancer progression and metastasis. Owing to restricted tools used to underpin the study of tumor invasion process, an on-site platform was developed to monitor this event in vitro. We used interdigital gold electrodes to monitor the dynamic process of cancer cells invading into extracellular matrix in situ continuously. Influences of collagen concentration and number of cancer cells on the measured impedance was exhibited. In addition, the parameters used to demonstrate the experiment results were optimized. The change of impedance magnitude indicated the cell-matrix interaction during invasion process. The potential further use of this platform would be complementary in cell studies when concerning metastasis.