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Besides the standard parameters used for colorectal cancer (CRC) management, new features are needed in clinical practice to improve progression-free and overall survival. In some cancers, the microenvironment mechanical properties can contribute to cancer progression and metastasis formation, or constitute a physical barrier for drug penetration or immune cell infiltration. These mechanical properties remain poorly known for colon tissues. Using a multidisciplinary approach including clinical data, physics and geostatistics, we characterized the stiffness of healthy and malignant colon specimens. For this purpose, we analyzed a prospective cohort of 18 patients with untreated colon adenocarcinoma using atomic force microscopy to generate micrometer-scale mechanical maps. We characterized the stiffness of normal epithelium samples taken far away or close to the tumor area and selected tumor tissue areas. These data showed that normal epithelium was softer than tumors. In tumors, stroma areas were stiffer than malignant epithelial cell areas. Among the clinical parameters, tumor left location, higher stage, and RAS mutations were associated with increased tissue stiffness. Thus, in patients with CRC, measuring tumor tissue rigidity may have a translational value and an impact on patient care., (© 2022. The Author(s).)

Tetraspanins are a family of transmembrane proteins that form a network of protein-protein interactions within the plasma membrane. Within this network, tetraspanin are thought to control the lateral segregation of their partners at the plasma membrane through mechanisms involving specific lipids. Here, we used a single molecule tracking approach to study the membrane behavior of tetraspanins in mammary epithelial cells and demonstrate that despite a common overall behavior, each tetraspanin (CD9, CD81 and CD82) has a specific signature in terms of dynamics. Furthermore, we demonstrated that tetraspanin dynamics on the cell surface are dependent on gangliosides. More specifically, we found that CD82 expression increases the dynamics of CD81 and alters its localization at the plasma membrane, this has no effect on the behavior of CD9. Our results provide new information on the ability of CD82 and gangliosides to differentially modulate the dynamics and organization of tetraspanins at the plasma membrane and highlight that its lipid and protein composition is involved in the dynamical architecture of the tetraspanin web. We predict that CD82 may act as a regulator of the lateral segregation of specific tetraspanins at the plasma membrane while gangliosides could play a crucial role in establishing tetraspanin-enriched areas.

The plasma membrane is a key actor of cell migration. For instance, its tension controls persistent cell migration and cell surface caveolae integrity. Then, caveolae constituents such as caveolin-1 can initiate a mechanotransduction loop that involves actin- and focal adhesion-dependent control of the mechanosensor YAP to finely tune cell migration. Tetraspanin CD82 (also named KAI-1) is an integral membrane protein and a metastasis suppressor. Its expression is lost in many cancers including breast cancer. It is a strong inhibitor of cell migration by a little-known mechanism. We demonstrated here that CD82 controls persistent 2D migration of EGF-induced single cells, stress fibers and focal adhesion sizes and dynamics. Mechanistically, we found that CD82 regulates membrane tension, cell surface caveolae abundance and YAP nuclear translocation in a caveolin-1-dependent manner. Altogether, our data show that CD82 controls 2D cell migration using membrane-driven mechanics involving caveolin and the YAP pathway.

ERBB2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2) amplification is associated with invasive breast cancer. We discovered that TOM1L1 (target of myb1-like 1) and ERBB2 co-amplification defines a novel mechanism involved in breast cancer metastatic progression. Upregulation of the vesicular trafficking protein TOM1L1 enhances plasma membrane delivery of membrane-type 1 matrix metalloprotease (MT1-MMP) for efficient extracellular matrix degradation and tumor cell dissemination.

Compartmentalization of Src tyrosine kinases (SFK) plays an important role in signal transduction induced by a number of extracellular stimuli. For example, Src mitogenic signaling induced by platelet-derived growth factor (PDGF) is initiated in cholesterol-enriched microdomain caveolae. How this Src subcellular localization is regulated is largely unknown. Here we show that the Tom1L1-clathrin heavy chain (CHC) complex negatively regulates the level of SFK in caveolae needed for the induction of DNA synthesis. Tom1L1 is both an interactor and a substrate of SFK. Intriguingly, it stimulates Src activity without promoting mitogenic signaling. We found that, upon association with CHC, Tom1L1 reduced the level of SFK in caveolae, thereby preventing its association with the PDGF receptor, which is required for the induction of mitogenesis. Similarly, the Tom1L1-CHC complex reduced also the level of oncogenic Src in cholesterol-enriched microdomains, thus affecting both its capacity to induce DNA synthesis and cell transformation. Conversely, Tom1L1, when not associated with CHC, accumulated in caveolae and promoted Src-driven DNA synthesis. We concluded that the Tom1L1-CHC complex defines a novel mechanism involved in negative regulation of mitogenic and transforming signals, by modulating SFK partitioning at the plasma membrane.

Gelsolin and calponin are well-characterized cytoskeletal proteins that are abundant and widely expressed in vertebrate tissues. It is also becoming apparent, however, that they are involved in cell signalling. In the present study, we show that gelsolin and calponin interact directly to form a high-affinity (K(d)=16 nM) 1:1 complex, by the use of fluorescent probes attached to both proteins, by affinity chromatography and by immunoprecipitation. These methods show that gelsolin can form high-affinity complexes with two calponin isoforms (basic h1 and acidic h3). They also show that gelsolin binds calponin through regions that have been identified previously as being calponin's actin-binding sites. Moreover, gelsolin does not interact with calponin while calponin is bound to F-actin. Reciprocal experiments to find calponin-binding sites on gelsolin show that these are in both the N- and C-terminal halves of gelsolin. Calponin has minimal effects on actin severing by gelsolin. In contrast, calponin markedly affects the nucleation activity of gelsolin. The maximum inhibition of nucleation by gelsolin was 50%, which was achieved with a ratio of two calponins for every gelsolin. Thus the interaction of calponin with gelsolin may play a regulatory role in the formation of actin filaments through modulation of gelsolin's actin-binding function and through the prevention of calponin's actin-binding activities.

The phosphorylation-dephosphorylation of serine and threonine residues of calponin is known to modulate in vitro its interaction with F-actin and is thought to regulate several biological processes in cells, involving either of the calponin isoforms. Here, we identify, for the first time, tyrosine-phosphorylated calponin h3 within COS 7 cells, before and after their transfection with the pSV vector containing cDNA encoding the cytoplasmic, Src-related, tyrosine kinase, Fyn. We then describe the specific tyrosine phosphorylation in vitro of calponin h1 and calponin h3 by this kinase. 32P-labeling of tyrosine residues was monitored by combined autoradiography, immunoblotting with a specific phosphotyrosine monoclonal antibody and dephosphorylation with the phosphotyrosine-specific protein phosphatase, YOP. PhosphorImager analyses showed the incorporation of maximally 1.4 and 2.0 mol of 32P per mol of calponin h3 and calponin h1, respectively. As a result, 75% and 68%, respectively, of binding to F-actin was lost by the phosphorylated calponins. Furthermore, F-actin, added at a two- or 10-fold molar excess, did not protect, but rather increased, the extent of 32P-labeling in both calponins. Structural analysis of the tryptic phosphopeptides from each 32P-labeled calponin revealed a single, major 32P-peptide in calponin h3, with Tyr261 as the phosphorylation site. Tyr261 was also phosphorylated in calponin h1, together with Tyr182. Collectively, the data point to the potential involvement, at least in living nonmuscle cells, of tyrosine protein kinases and the conserved Tyr261, located in the third repeat motif of the calponin molecule, in a new level of regulation of the actin-calponin interaction.

The cytoplasmic tyrosine kinase cSrc is involved in the regulation of many important cellular functions including cell growth and transformation, and its activity is down-regulated by phosphorylation of the Tyr530 residue by the COOH-terminal Src tyrosine kinase, Csk. Because cSrc was previously found overexpressed, activated, and in some cases mutated in carcinoma, we investigated whether it could act as a tumor antigen. We show that whereas no autoantibodies were found against cSrc or its relative Fyn, up to 20% of patients with carcinoma had high-affinity autoantibodies against Csk. Immunity mainly resulted from a secondary response, as indicated by the presence of IgG1 in the sera. Antibodies were linked to the cancer because they were not detected in healthy subjects nor in patients with unrelated diseases, and their levels decreased in the sera of patients after surgical resection. Furthermore, they behaved as early markers of epithelial transformation because they were present in sera of patients with early-stage tumors and precancerous lesions such as colorectal polyps and in sera of patients that were scored negative for other cancer serological markers (CEA, CA15-3, CA19-9, p53 antibodies). Finally the presence of these antibodies was attributed, at least in part, to a substantial elevation of Csk protein levels in the corresponding tumors. However a strong increase in Src activity was also observed in these tissues, which suggested that Csk cannot regulate Src-like activity in carcinoma. Taken together, these data demonstrate that Csk acts as an autoantigen, and the detection of anti-Csk antibodies may have potential diagnostic usefulness in the early detection and postoperative follow-up of patients with carcinoma.

We have previously shown an important function of phosphatidylinositol 3-kinase (PI3K)alpha(p85alpha-p110alpha) and PI3Kbeta (p85-alpha-p110beta) for DNA synthesis induced by various mitogens in non transformed fibroblasts and we now report a specific role of these enzymes in human colon cancer cell growth. Using antibodies specific to p110alpha and to p110beta catalytic subunits, increase in PI3Kalpha and PI3Kbeta activities was detected in 15/19 human tumour biopsies relative to adjacent normal mucosa of human colon and bladder. Increase in such activities was also observed in adenocarcinoma cell lines CaCo2, CO115, HCT 116, LS 174T and WiDr relative to non-transformed fibroblasts. Maximal PI3Kalpha activity was observed for LS 174T and PI3Kbeta activity for WiDr cells. This was partly correlated with an increase in p110alpha and p110beta protein levels both in some primary tumours and established cell lines, suggesting that PI3K overexpression is involved in enzymatic deregulation. Functional consequence of such activation was assessed by a microinjection approach. An injection of neutralizing antibody specific to p110beta in WiDr, HCT116 and CO 115 cells inhibited de novo DNA synthesis, whereas antibodies specific to p110gamma had no effect. Neutralizing antibodies specific to p110alpha induced apoptosis, a response that was reverted by treating cells with the caspase inhibitor z-VAD-fmk. However anti-p110beta and anti-p110gamma antibodies did not affect cell survival. We concluded that PI3Kalpha and PI3Kbeta play important roles in human colon cancer cell growth with a specific function for PI3Kbeta in de novo DNA synthesis and an involvement of PI3Kalpha in cell survival.

In cold-acclimated (CA) birds, lipids play a crucial role in regulatory thermogenesis by acting both as substrates for and activators of thermogenic processes. The capacity to supply lipids to thermogenic tissues, which could limit cold thermogenesis, was assessed in CA ducklings (5 wk old, 4 degrees C) and compared with thermoneutral controls (TN, 25 degrees C). In CA ducklings, basal lipolytic activity of adipose tissue fragments was higher (202 +/- 9 vs. 130 +/- 14 nmol glycerol released . 100 mg tissue-1 . h-1, +55%) than in TN controls, while glucagon had a much higher stimulatory effect (+140 to +500% depending on dose). This was consistent with increased plasma levels of nonesterified fatty acids (FA, +57%) and glycerol (+31%) in vivo. In vitro endothelial lipase activity per organ was higher in CA than in TN ducklings in red gastrocnemius muscle (6.3 +/- 0.6 vs. 3.5 +/- 0.3 microeq nonesterified FA released per hour, +80%) and liver (+55%). The intracellular FA-binding capacity of (12-18 kDa) proteins was higher in gastrocnemius muscle (+43%) and liver (+74%) from CA ducklings. In gastrocnemius, it was linked to a higher content (21 +/- 2 vs. 15 +/- 2 microg/mg protein, +37%) of an intracellular 15.4-kDa FA-binding protein. These in vitro results indicate that coordinated increases in FA supply from adipose tissue, cellular uptake of lipoprotein-derived FA, and intracellular FA transport capacity occur in CA ducklings endowed with higher thermogenic capacity and cold endurance.

To enlighten the mechanism of inhibition of prostacyclin (PGI2) production by n-3 fatty acids, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, cultured endothelial cells were incubated with albumin bound-EPA or -DHA for 22 h. Under these conditions, PGI2 formation in response to bradykinin, calcium ionophore or exogenous arachidonic acid was equally inhibited by 50%, suggesting that the inhibition might occur downstream the phospholipase step, likely at the level of PGH synthase and/or PGI2 synthase activities. Western blot analysis indicated that the mass of the constitutive isoform of PGH synthase (PGH synthase 1), but not PGI2 synthase, was significantly reduced in n-3 fatty acid-enriched cells. In subsequent experiments, PGH synthase 1 mRNA level, measured by northern blotting, was also decreased in n-3 supplemented cells. This reduction was not due to mRNA destabilization. None of these parameters were altered by similar enrichment with oleic acid (OA). These results suggest that EPA and DHA may affect PGH synthase 1 expression, presumably at the transcriptional level.

The cross-reactivity of the PGI3 metabolite, delta 17-6-keto-PGF1 alpha, with antibodies against 6-keto-PGF1 alpha for radioimmunoassays (RIA) has been investigated. Delta 17-6-keto-PGF1 alpha was obtained either from commercial sources or after its purification from endothelial cells. In the latter case, primary cultured bovine aortic endothelial cells were incubated for 20 min at 37 degrees C with 10 microM eicosapentaenoic acid (EPA) in the presence of 2 microM 13-hydroperoxy-octadecadienoic acid, and activator of the EPA cyclooxygenation, and the 6-keto-PGF1 alpha and beta 17-6-keto-PGF1 alpha produced were separated by RP-HPLC. Then, cross-reactivities of the commercial and purified beta 17-6-keto-PGF1 alpha with 6-keto-PGF1 alpha antibodies were determined and found not to exceed 10%. In addition, the amounts of prostacyclin-related compounds detected by direct measurements in media of cells loaded with EPA were compared with those obtained after purification of 6-keto-PGF1 alpha. In accordance with the cross-reactivity data, we found that RIA in media mainly measured 6-keto-PGF1 alpha, the beta 17-6-keto-PGF1 alpha formed being undetected at 90%. It is concluded that 6-keto-PGF1 alpha antibodies generally used for RIA of 6-keto-PGF1 alpha are highly specific since they can discriminate a metabolite bearing an additional double band such as the PGI3 metabolite beta 17-6-keto-PGF1 alpha.

The anti-aggregatory activity of endothelial cells being affected by eicosapentaenoic (EPA, 20:5(n-3)) and docosahexaenoic (DHA, 22:6(n-3)) acids, the two main polyunsaturated fatty acids of fish oil, these fatty acids, as well as their intermediary, docosapentaenoic acid (DPA, 22:5(n-3)), were investigated with respect to their metabolism. Primary cultured bovine aortic endothelial cells were supplemented for 22 h at 37 degrees C with either n-3 fatty acid, and the fatty acids of cell media, of cell lipid classes, and of choline and ethanolamine glycerophospholipids (PC and PE) were quantified. Endothelial cells converted each of the three fatty acids into the two others. They were found esterified in cell lipids and partly released in cell media, the respective parts varying according to the fatty acid. For instance, half of the DPA formed from EPA and two third of the EPA formed from DPA were released in the media. Moreover, the DHA formed from EPA and DPA was not esterified but released in media. In addition, the esterified counterparts were found in either PC or PE, depending on whether they were added or formed by conversions. It is concluded that EPA, DPA and DHA are actively interconverted each others, and differ substantially in terms of distribution between media and cells, and within phospholipid classes.

S-adenosyl-l-methionine (AdoMet) has been reported to affect events linked to noradrenergic neurotransmission. In the present work, we studied the effect of AdoMet on norepinephrine (NE)-stimulated inositol phosphate production in 3H-inositol-labelled crude synaptosomal suspensions of rat brain. AdoMet (50-1000 microM) decreased both the synthesis of labelled polyphosphoinositide (30-50%) and the release of inositol mono- and bisphosphate (40-50%). The AdoMet effect was not dependent on NE concentration (10-1000 microM), suggesting that the inhibition of inositol phosphate release was not the result of a modification of the norepinephrine binding to its receptor sites. S-adenosyl-L-homocysteine (AdoHcy) (1 mM) an inhibitor of methyltransferase activities, partially inhibited (70%) the AdoMet (0.1 mM) effect, indicating that the methylation processes cannot explain all the effects observed. We conclude that, in addition to previously reported effects of AdoMet on NE transport, AdoMet may reduce NE-linked intracellular signalling.