Mitochondrial HER2 stimulates respiration and promotes tumorigenicity.

BACKGROUND: Amplification of HER2, a receptor tyrosine kinase and a breast cancer-linked oncogene, is associated with aggressive disease. HER2 protein is localised mostly at the cell membrane, but a fraction translocates to mitochondria. Whether and how mitochondrial HER2 contributes to tumorigenicity is currently unknown. METHODS: We enriched the mitochondrial (mt-)HER2 fraction in breast cancer cells using an N-terminal mitochondrial targeting sequence and analysed how this manipulation impacts bioenergetics and tumorigenic properties. The role of the tyrosine kinase activity of mt-HER2 was assessed in wild type, kinase-dead (K753M) and kinase-enhanced (V659E) mtHER2 constructs. RESULTS: We document that mt-HER2 associates with the oxidative phosphorylation system, stimulates bioenergetics and promotes larger respiratory supercomplexes. mt-HER2 enhances proliferation and invasiveness in vitro and tumour growth and metastatic potential in vivo, in a kinase activity-dependent manner. On the other hand, constitutively active mt-HER2 provokes excessive mitochondria ROS generation, sensitises to cell death, and restricts growth of primary tumours, suggesting that regulation of HER2 activity in mitochondria is required for the maximal pro-tumorigenic effect. CONCLUSIONS: mt-HER2 promotes tumorigenicity by supporting bioenergetics and optimal redox balance.

A homozygous stop-gain variant in ARHGAP42 is associated with childhood interstitial lung disease, systemic hypertension, and immunological findings.

ARHGAP42 encodes Rho GTPase activating protein 42 that belongs to a member of the GTPase Regulator Associated with Focal Adhesion Kinase (GRAF) family. ARHGAP42 is involved in blood pressure control by regulating vascular tone. Despite these findings, disorders of human variants in the coding part of ARHGAP42 have not been reported. Here, we describe an 8-year-old girl with childhood interstitial lung disease (chILD), systemic hypertension, and immunological findings who carries a homozygous stop-gain variant (c.469G>T, p.(Glu157Ter)) in the ARHGAP42 gene. The family history is notable for both parents with hypertension. Histopathological examination of the proband lung biopsy showed increased mural smooth muscle in small airways and alveolar septa, and concentric medial hypertrophy in pulmonary arteries. ARHGAP42 stop-gain variant in the proband leads to exon 5 skipping, and reduced ARHGAP42 levels, which was associated with enhanced RhoA and Cdc42 expression. This is the first report linking a homozygous stop-gain variant in ARHGAP42 with a chILD disorder, systemic hypertension, and immunological findings in human patient. Evidence of smooth muscle hypertrophy on lung biopsy and an increase in RhoA/ROCK signaling in patient cells suggests the potential mechanistic link between ARHGAP42 deficiency and the development of chILD disorder.

Cytoplasmic Tail of MT1-MMP: A Hub of MT1-MMP Regulation and Function.

MT1-MMP (MMP-14) is a multifunctional protease that regulates ECM degradation, activation of other proteases, and a variety of cellular processes, including migration and viability in physiological and pathological contexts. Both the localization and signal transduction capabilities of MT1-MMP are dependent on its cytoplasmic domain that constitutes the final 20 C-terminal amino acids, while the rest of the protease is extracellular. In this review, we summarize the ways in which the cytoplasmic tail is involved in regulating and enacting the functions of MT1-MMP. We also provide an overview of known interactors of the MT1-MMP cytoplasmic tail and the functional significance of these interactions, as well as further insight into the mechanisms of cellular adhesion and invasion that are regulated by the cytoplasmic tail.

Autoimmunity, cancer and COVID-19 abnormally activate wound healing pathways: critical role of inflammation.

Recent evidence indicates that targeting IL-6 provides broad therapeutic approaches to several diseases. In patients with cancer, autoimmune diseases, severe respiratory infections [e.g. coronavirus disease 2019 (COVID-19)] and wound healing, IL-6 plays a critical role in modulating the systemic and local microenvironment. Elevated serum levels of IL-6 interfere with the systemic immune response and are associated with disease progression and prognosis. As already noted, monoclonal antibodies blocking either IL-6 or binding of IL-6 to receptors have been used/tested successfully in the treatment of rheumatoid arthritis, many cancer types, and COVID-19. Therefore, in the present review, we compare the impact of IL-6 and anti-IL-6 therapy to demonstrate common (pathological) features of the studied diseases such as formation of granulation tissue with the presence of myofibroblasts and deposition of new extracellular matrix. We also discuss abnormal activation of other wound-healing-related pathways that have been implicated in autoimmune disorders, cancer or COVID-19.

Exosomes produced by melanoma cells significantly influence the biological properties of normal and cancer-associated fibroblasts.

The incidence of cutaneous malignant melanoma is increasing worldwide. While the treatment of initial stages of the disease is simple, the advanced disease frequently remains fatal despite novel therapeutic options . This requires identification of novel therapeutic targets in melanoma. Similarly to other types of tumours, the cancer microenvironment plays a prominent role and determines the biological properties of melanoma. Importantly, melanoma cell-produced exosomes represent an important tool of intercellular communication within this cancer ecosystem. We have focused on potential differences in the activity of exosomes produced by melanoma cells towards melanoma-associated fibroblasts and normal dermal fibroblasts. Cancer-associated fibroblasts were activated by the melanoma cell-produced exosomes significantly more than their normal counterparts, as assessed by increased transcription of genes for inflammation-supporting cytokines and chemokines, namely IL-6 or IL-8. We have observed that the response is dependent on the duration of the stimulus via exosomes and also on the quantity of exosomes. Our study demonstrates that melanoma-produced exosomes significantly stimulate the tumour-promoting proinflammatory activity of cancer-associated fibroblasts. This may represent a potential new target of oncologic therapy .

Cancer-Associated Fibroblasts Influence the Biological Properties of Malignant Tumours via Paracrine Secretion and Exosome Production.

Cancer-associated fibroblasts (CAFs) are an essential component of the tumour microenvironment. They represent a heterogeneous group of cells that are under the control of cancer cells and can reversely influence the cancer cell population. They affect the cancer cell differentiation status, and the migration and formation of metastases. This is achieved through the production of the extracellular matrix and numerous bioactive factors. IL-6 seems to play the central role in the communication of noncancerous and cancer cells in the tumour. This review outlines the role of exosomes in cancer cells and cancer-associated fibroblasts. Available data on the exosomal cargo, which can significantly intensify interactions in the tumour, are summarised. The role of exosomes as mediators of the dialogue between cancer cells and cancer-associated fibroblasts is discussed together with their therapeutic relevance. The functional unity of the paracrine- and exosome-mediated communication of cancer cells with the tumour microenvironment represented by CAFs is worthy of attention.

Thermo- and ROS-Responsive Self-Assembled Polymer Nanoparticle Tracers for 19F MRI Theranostics.

Fluorine-19 magnetic resonance imaging (19F MRI) enables detailed in vivo tracking of fluorine-containing tracers and is therefore becoming a particularly useful tool in noninvasive medical imaging. In previous studies, we introduced biocompatible polymers based on the hydrophilic monomer N-(2-hydroxypropyl)methacrylamide (HPMA) and the thermoresponsive monomer N-(2,2-difluoroethyl)acrylamide (DFEA). These polymers have abundant magnetically equivalent fluorine atoms and advantageous properties as 19F MRI tracers. Furthermore, in this pilot study, we modified these polymers by introducing a redox-responsive monomer. As a result, our polymers changed their physicochemical properties once exposed to an oxidative environment. Reactive oxygen species (ROS)-responsive polymers were prepared by incorporating small amounts (0.9-4.5 mol %) of the N-[2-(ferrocenylcarboxamido)ethyl]acrylamide (FcCEA) monomer, which is hydrophobic and diamagnetic in the reduced electroneutral (Fe(II), ferrocene) state but hydrophilic and paramagnetic in the oxidized (Fe(III), ferrocenium cation) state. This property can be useful for theranostic purposes (therapy and diagnostic purposes), especially, in terms of ROS-responsive drug-delivery systems. In the reduced state, these nanoparticles remain self-assembled with the encapsulated drug but release the drug upon oxidation in ROS-rich tumors or inflamed tissues.

Invadopodia Structure in 3D Environment Resolved by Near-Infrared Branding Protocol Combining Correlative Confocal and FIB-SEM Microscopy.

Cancer cell invasion through tissue barriers is the intrinsic feature of metastasis, the most life-threatening aspect of cancer. Detailed observation and analysis of cancer cell behaviour in a 3D environment is essential for a full understanding of the mechanisms of cancer cell invasion. The inherent limits of optical microscopy resolution do not allow to for in-depth observation of intracellular structures, such as invadopodia of invading cancer cells. The required resolution can be achieved using electron microscopy techniques such as FIB-SEM. However, visualising cells in a 3D matrix using FIB-SEM is challenging due to difficulties with localisation of a specific cell deep within the resin block. We have developed a new protocol based on the near-infrared branding (NIRB) procedure that extends the pattern from the surface grid deep inside the resin. This 3D burned pattern allows for precise trimming followed by targeted 3D FIB-SEM. Here we present detailed 3D CLEM results combining confocal and FIB-SEM imaging of cancer cell invadopodia that extend deep into the collagen meshwork.

Estrogen Receptor Modulators in Viral Infections Such as SARS-CoV-2: Therapeutic Consequences.

COVID-19 is a pandemic respiratory disease caused by the SARS-CoV-2 coronavirus. The worldwide epidemiologic data showed higher mortality in males compared to females, suggesting a hypothesis about the protective effect of estrogens against severe disease progression with the ultimate end being patient's death. This article summarizes the current knowledge regarding the potential effect of estrogens and other modulators of estrogen receptors on COVID-19. While estrogen receptor activation shows complex effects on the patient's organism, such as an influence on the cardiovascular/pulmonary/immune system which includes lower production of cytokines responsible for the cytokine storm, the receptor-independent effects directly inhibits viral replication. Furthermore, it inhibits the interaction of IL-6 with its receptor complex. Interestingly, in addition to natural hormones, phytestrogens and even synthetic molecules are able to interact with the estrogen receptor and exhibit some anti-COVID-19 activity. From this point of view, estrogen receptor modulators have the potential to be included in the anti-COVID-19 therapeutic arsenal.

A Screen for PKN3 Substrates Reveals an Activating Phosphorylation of ARHGAP18.

Protein kinase N3 (PKN3) is a serine/threonine kinase implicated in tumor progression of multiple cancer types, however, its substrates and effector proteins still remain largely understudied. In the present work we aimed to identify novel PKN3 substrates in a phosphoproteomic screen using analog sensitive PKN3. Among the identified putative substrates we selected ARHGAP18, a protein from RhoGAP family, for validation of the screen and further study. We confirmed that PKN3 can phosphorylate ARHGAP18 in vitro and we also characterized the interaction of the two proteins, which is mediated via the N-terminal part of ARHGAP18. We present strong evidence that PKN3-ARHGAP18 interaction is increased upon ARHGAP18 phosphorylation and that the phosphorylation of ARHGAP18 by PKN3 enhances its GAP domain activity and contributes to negative regulation of active RhoA. Taken together, we identified new set of potential PKN3 substrates and revealed a new negative feedback regulatory mechanism of Rho signaling mediated by PKN3-induced ARHGAP18 activation.

Interleukin-6: Molecule in the Intersection of Cancer, Ageing and COVID-19.

Interleukin-6 (IL-6) is a cytokine with multifaceted effects playing a remarkable role in the initiation of the immune response. The increased level of this cytokine in the elderly seems to be associated with the chronic inflammatory setting of the microenvironment in aged individuals. IL-6 also represents one of the main signals in communication between cancer cells and their non-malignant neighbours within the tumour niche. IL-6 also participates in the development of a premetastatic niche and in the adjustment of the metabolism in terminal-stage patients suffering from a malignant disease. IL-6 is a fundamental factor of the cytokine storm in patients with severe COVID-19, where it is responsible for the fatal outcome of the disease. A better understanding of the role of IL-6 under physiological as well as pathological conditions and the preparation of new strategies for the therapeutic control of the IL-6 axis may help to manage the problems associated with the elderly, cancer, and serious viral infections.

Solid cancer: the new tumour spread endpoint opens novel opportunities.

Novel androgen deprivation agents delay metastasis in non-metastatic, castration-resistant, prostate cancer (nmCRPC). The recent regulatory guidance: considerations for metastasis-free survival endpoint in clinical trials, opens new opportunities in cell biology, medicinal chemistry and advanced imaging. Past failures are the likely result of equating tumour shrinkage to efficacy, rather than inhibition of tumour spread. In the future, the selection of anti-metastasis drug candidates will probably be based on anti-migratory rather than anti-proliferative potential. Oligometastatic cancer coupled with advanced imaging can serve as a clinical proof-of-concept model.

ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility.

The tyrosine kinase Src acts as a key regulator of cell motility by phosphorylating multiple protein substrates that control cytoskeletal and adhesion dynamics. In an earlier phosphotyrosine proteomics study, we identified a novel Rho-GTPase activating protein, now known as ARHGAP42, as a likely biologically relevant Src substrate. ARHGAP42 is a member of a family of RhoGAPs distinguished by tandem BAR-PH domains lying N-terminal to the GAP domain. Like other family members, ARHGAP42 acts preferentially as a GAP for RhoA. We show that Src principally phosphorylates ARHGAP42 on tyrosine 376 (Tyr-376) in the short linker between the BAR-PH and GAP domains. The expression of ARHGAP42 variants in mammalian cells was used to elucidate its regulation. We found that the BAR domain is inhibitory toward the GAP activity of ARHGAP42, such that BAR domain deletion resulted in decreased active GTP-bound RhoA and increased cell motility. With the BAR domain intact, ARHGAP42 GAP activity could be activated by phosphorylation of Tyr-376 to promote motile cell behavior. Thus, phosphorylation of ARHGAP42 Tyr-376 is revealed as a novel regulatory event by which Src can affect actin dynamics through RhoA inhibition.

6-Substituted purines as ROCK inhibitors with anti-metastatic activity.

Rho-associated serine/threonine kinases (ROCKs) are principal regulators of the actin cytoskeleton that regulate the contractility, shape, motility, and invasion of cells. We explored the relationships between structure and anti-ROCK2 activity in a group of purine derivatives substituted at the C6 atom by piperidin-1-yl or azepan-1-yl groups. Structure-activity relationship (SAR) analyses suggested that anti-ROCK activity is retained, and may be further increased, by substitution of the parent compounds at the C2 atom or by expansion of the C6 side chain. These inhibitors of ROCK can reach effective concentrations within cells, as demonstrated by a decrease in phosphorylation of the ROCK target MLC, and by inhibition of the ROCK-dependent invasion of melanoma cells in the collagen matrix. Our study may be useful for further optimization of C6-substituted purine inhibitors of ROCKs and of other sensitive kinases identified by the screening of a broad panel of protein kinases.

Fibroblasts potentiate melanoma cells in vitro invasiveness induced by UV-irradiated keratinocytes.

Melanoma represents a malignant disease with steadily increasing incidence. UV-irradiation is a recognized key factor in melanoma initiation. Therefore, the efficient prevention of UV tissue damage bears a critical potential for melanoma prevention. In this study, we tested the effect of UV irradiation of normal keratinocytes and their consequent interaction with normal and cancer-associated fibroblasts isolated from melanoma, respectively. Using this model of UV influenced microenvironment, we measured melanoma cell migration in 3-D collagen gels. These interactions were studied using DNA microarray technology, immunofluorescence staining, single cell electrophoresis assay, viability (dead/life) cell detection methods, and migration analysis. We observed that three 10 mJ/cm2 fractions at equal intervals over 72 h applied on keratinocytes lead to a 50% increase (p < 0.05) in in vitro invasion of melanoma cells. The introduction cancer-associated fibroblasts to such model further significantly stimulated melanoma cells in vitro invasiveness to a higher extent than normal fibroblasts. A panel of candidate gene products responsible for facilitation of melanoma cells invasion was defined with emphasis on IL-6, IL-8, and CXCL-1. In conclusion, this study demonstrates a synergistic effect between cancer microenvironment and UV irradiation in melanoma invasiveness under in vitro condition.

Simultaneous blocking of IL-6 and IL-8 is sufficient to fully inhibit CAF-induced human melanoma cell invasiveness.

Tumour microenvironment plays a critical role in cell invasion and metastasis. To investigate the role of cancer-associated fibroblasts (CAFs) in melanoma cell invasiveness, we used 3D spheroid invasion assay. The effect of conditioned media from normal fibroblasts and CAFs cultivated alone or co-cultivated with melanoma cells on BLM or A2058 melanoma spheroid invasion was analysed. We found that conditioned media from CAFs and CAFs co-cultured with melanoma cells, especially, promote invasion and migration, without significant effect on melanoma cell proliferation. We further analysed the expression of pro-invasive cytokines IL-8 and IL-6 in media and found that melanoma cells are dominant producers of IL-8 and fibroblasts are dominant producers of IL-6 in 2D monocultures, while co-cultivation of CAFs with melanoma cells induces production/secretion of IL-6 and IL-8 into the media. The analyses of IL-6 levels in 3D cultures and human melanoma samples, however, revealed that at least in some cases IL-6 is also produced directly by melanoma cells. Analysis of the role of IL-6 and IL-8 in CAF-induced melanoma invasion, using neutralising antibodies, revealed that simultaneous blocking of IL-6 and IL-8 is sufficient to fully inhibit CAF-induced human melanoma cell invasiveness. In summary, these experiments indicate the important role of CAFs and IL-8 and IL-6 cytokines in melanoma cell invasiveness.

PKCα promotes the mesenchymal to amoeboid transition and increases cancer cell invasiveness.

BACKGROUND: The local invasion of tumor cells into the surrounding tissue is the first and most critical step of the metastatic cascade. Cells can invade either collectively, or individually. Individual cancer cell invasion can occur in the mesenchymal or amoeboid mode, which are mutually interchangeable. This plasticity of individual cancer cell invasiveness may represent an escape mechanism for invading cancer cells from anti-metastatic treatment. METHODS: To identify new signaling proteins involved in the plasticity of cancer cell invasiveness, we performed proteomic analysis of the amoeboid to mesenchymal transition with A375m2 melanoma cells in a 3D Matrigel matrix. RESULTS: In this screen we identified PKCα as an important protein for the maintenance of amoeboid morphology. We found that the activation of PKCα resulted in the mesenchymal-amoeboid transition of mesenchymal K2 and MDA-MB-231 cell lines. Consistently, PKCα inhibition led to the amoeboid-mesenchymal transition of amoeboid A375m2 cells. Next, we showed that PKCα inhibition resulted in a considerable decrease in the invading abilities of all analyzed cancer cell lines. CONCLUSIONS: Our results suggest that PKCα is an important protein for maintenance of the amoeboid morphology of cancer cells, and that downregulation of PKCα results in the amoeboid to mesenchymal transition. Our data also suggest that PKCα is important for both mesenchymal and amoeboid invasiveness, making it an attractive target for anti-metastatic therapies.

CAS directly interacts with vinculin to control mechanosensing and focal adhesion dynamics.

Focal adhesions are cellular structures through which both mechanical forces and regulatory signals are transmitted. Two focal adhesion-associated proteins, Crk-associated substrate (CAS) and vinculin, were both independently shown to be crucial for the ability of cells to transmit mechanical forces and to regulate cytoskeletal tension. Here, we identify a novel, direct binding interaction between CAS and vinculin. This interaction is mediated by the CAS SRC homology 3 domain and a proline-rich sequence in the hinge region of vinculin. We show that CAS localization in focal adhesions is partially dependent on vinculin, and that CAS-vinculin coupling is required for stretch-induced activation of CAS at the Y410 phosphorylation site. Moreover, CAS-vinculin binding significantly affects the dynamics of CAS and vinculin within focal adhesions as well as the size of focal adhesions. Finally, disruption of CAS binding to vinculin reduces cell stiffness and traction force generation. Taken together, these findings strongly implicate a crucial role of CAS-vinculin interaction in mechanosensing and focal adhesion dynamics.

Educate, not kill: treating cancer without triggering its defenses.

Traditionally, anticancer therapies focus on restraining uncontrolled proliferation. However, these cytotoxic therapies expose cancer cells to direct killing, instigating the process of natural selection favoring survival of resistant cells that become the foundation for tumor progression and therapy failure. Recognizing this phenomenon has prompted the development of alternative therapeutic strategies. Here we propose strategies targeting cancer hallmarks beyond proliferation, aiming at re-educating cancer cells towards a less malignant phenotype. These strategies include controlling cell dormancy, transdifferentiation therapy, normalizing the cancer microenvironment, and using migrastatic therapy. Adaptive resistance to these educative strategies does not confer a direct proliferative advantage to resistant cells, as non-resistant cells are not subject to eradication, thereby delaying or preventing the development of therapy-resistant tumors.