Trajectory Inference for Unraveling Dynamic Biological Processes from Raman Spectral Data.

Cell heterogeneity is a crucial parameter for understanding the complexity of numerous biomedical issues. Trajectory inference-based approaches are recent tools developed for single-cell transcriptomics (scRNA-seq) data analysis. They aim to reconstruct evolving pathways from the variety of cell states that coexist simultaneously in a cell population. We propose to expand this concept to Raman spectroscopy, a label-free modality that probes the global molecular nature of a sample, by investigating the dynamics of adipocyte differentiation.

Type I Collagen Aging Increases Expression and Activation of EGFR and Induces Resistance to Erlotinib in Lung Carcinoma in 3D Matrix Model.

Type I collagen is the major structural component of lung stroma. Because of its long half-life, type I collagen undergoes post-translational modifications such as glycation during aging process. These modifications have been shown to impact the structural organization of type I collagen fibers. In the present work we evaluated the impact of collagen aging on lung carcinoma cells response to erlotinib-induced cytotoxicity and apoptosis, and on Epidermal Growth Factor Receptor (EGFR) expression and phosphorylation. To this end, experiments were performed in 2D and 3D matrix models established from type I collagen extracted from adult (10 weeks-old) and old (100 weeks-old) rat's tail tendons. Our results show that old collagen induces a significant increase in EGFR expression and phosphorylation when compared to adult collagen in 3D matrix but not in 2D coating. Such modification was associated to an increase in the IC50 of erlotinib in the presence of old collagen and a lower sensitivity to drug-induced apoptosis. These data suggest that collagen aging confers resistance to the cytotoxic and apoptotic effects of therapies targeting EGFR kinase function in lung carcinoma. Moreover, our data underline the importance of the 3D matrix environment in this process.

DDR1 and MT1-MMP Expression Levels Are Determinant for Triggering BIK-Mediated Apoptosis by 3D Type I Collagen Matrix in Invasive Basal-Like Breast Carcinoma Cells.

Type I collagen is the major adhesive component in breast interstitial stroma, which represents the first barrier against tumor cell invasion after basement-membrane degradation. Among cellular receptors, type I collagen is able to activate discoidin domain receptors DDR1 and DDR2. We have previously shown that in 3D collagen matrix, DDR1 plays a key role as it promotes cell growth suppression and apoptosis through the upregulation of the pro-apoptotic mediator BIK in noninvasive luminal-like breast carcinoma cells. We have also shown that MT1-MMP is able to rescue these cells and protect them against the effects induced by collagen/DDR1/BIK axis. Our data suggested that the protective effect of MT1-MMP might be mediated through the degradation of type I collagen and/or DDR1 cleavage. Decreased DDR1 expression has been associated with the epithelial to mesenchymal transition process in breast cancer, and its overexpression in aggressive basal-like breast cancer cells reduces their invasiveness in 3D cultures and in vivo. In the present work, we propose to study the role of MT1-MMP in the resistance against collagen-induced apoptosis in basal-like breast carcinoma MDA-MB-231 cells. We aimed to investigate whether MT1-MMP depletion is able to restore apoptosis mediated by collagen/DDR1/BIK axis and to verify if such depletion is able to restore full-length DDR1 expression and phosphorylation. ShRNA strategy against MT1-MMP mRNA was able to partially restore full length DDR1 expression and phosphorylation. This was accompanied by a decrease in cell growth and an upregulation of BIK expression. This suggested that MT1-MMP expression in basal-like breast carcinoma cells, in addition to a low basal level of DDR1 expression, protects these cells against collagen-induced apoptosis via DDR1 cleavage. Since DDR1 was moderately expressed in MDA-MB-231 cells, we then investigated whether overexpression of DDR1 could be able to increase its ability to suppress cell growth and to induce apoptosis. Data showed that overexpression of DDR1 induced a decrease in cell growth and an increase in BIK expression, suggesting that moderate expression level of full length DDR1 in basal-like breast carcinoma provides them with a capacity to resist to collagen-induced cell growth suppression and apoptosis. Finally, the combined overexpression of DDR1 and depletion of MT1-MMP in MDA-MB-231 cells synergistically increased collagen-induced cell growth suppression and apoptosis to a level similar to that observed in luminal breast carcinoma. Taken together, our data suggest that during the acquisition of mesenchymal features, the low level of DDR1 expression should be considered as an important biomarker in the prognosis of basal-like breast carcinoma, conferring them a high rate of cell growth and resistance to BIK-mediated apoptosis induced by the stromal collagen.

Age-related modifications of type I collagen impair DDR1-induced apoptosis in non-invasive breast carcinoma cells.

Type I collagen and DDR1 axis has been described to decrease cell proliferation and to initiate apoptosis in non-invasive breast carcinoma in three-dimensional cell culture matrices. Moreover, MT1-MMP down-regulates these effects. Here, we address the effect of type I collagen aging and MT1-MMP expression on cell proliferation suppression and induced-apoptosis in non-invasive MCF-7 and ZR-75-1 breast carcinoma. We provide evidence for a decrease in cell growth and an increase in apoptosis in the presence of adult collagen when compared to old collagen. This effect involves a differential activation of DDR1, as evidenced by a higher DDR1 phosphorylation level in adult collagen. In adult collagen, inhibition of DDR1 expression and kinase function induced an increase in cell growth to a level similar to that observed in old collagen. The impact of aging on the sensitivity of collagen to MT1-MMP has been reported recently. We used the MT1-MMP expression strategy to verify whether, by degrading adult type I collagen, it could lead to the same phenotype observed in old collagen 3D matrix. MT1-MMP overexpression abrogated the proliferation suppression and induced-apoptosis effects only in the presence of adult collagen. This suggests that differential collagen degradation by MT1-MMP induced a structural disorganization of adult collagen and inhibits DDR1 activation. This could in turn impair DDR1-induced cell growth suppression and apoptosis. Taken together, our data suggest that modifications of collagen structural organization, due to aging, contribute to the loss of the growth suppression and induced apoptosis effect of collagen in luminal breast carcinoma. MT1-MMP-dependent degradation and aging of collagen have no additive effects on these processes.

Corrigendum: Discoidin Domain Receptors: Potential Actors and Targets in Cancer.

[This corrects the article on p. 55 in vol. 7, PMID: 27014069.].

Type I collagen aging impairs discoidin domain receptor 2-mediated tumor cell growth suppression.

Tumor cells are confronted to a type I collagen rich environment which regulates cell proliferation and invasion. Biological aging has been associated with structural changes of type I collagen. Here, we address the effect of collagen aging on cell proliferation in a three-dimensional context (3D).We provide evidence for an inhibitory effect of adult collagen, but not of the old one, on proliferation of human fibrosarcoma HT-1080 cells. This effect involves both the activation of the tyrosine kinase Discoidin Domain Receptor 2 (DDR2) and the tyrosine phosphatase SHP-2. DDR2 and SHP-2 were less activated in old collagen. DDR2 inhibition decreased SHP-2 phosphorylation in adult collagen and increased cell proliferation to a level similar to that observed in old collagen.In the presence of old collagen, a high level of JAK2 and ERK1/2 phosphorylation was observed while expression of the cell cycle negative regulator p21CIP1 was decreased. Inhibition of DDR2 kinase function also led to an increase in ERK1/2 phosphorylation and a decrease in p21CIP1 expression. Similar signaling profile was observed when DDR2 was inhibited in adult collagen. Altogether, these data suggest that biological collagen aging could increase tumor cell proliferation by reducingthe activation of the key matrix sensor DDR2.

Discoidin Domain Receptors: Potential Actors and Targets in Cancer.

The extracellular matrix critically controls cancer cell behavior by inducing several signaling pathways through cell membrane receptors. Besides conferring structural properties to tissues around the tumor, the extracellular matrix is able to regulate cell proliferation, survival, migration, and invasion. Among these receptors, the integrins family constitutes a major class of receptors that mediate cell interactions with extracellular matrix components. Twenty years ago, a new class of extracellular matrix receptors has been discovered. These tyrosine kinase receptors are the two discoidin domain receptors DDR1 and DDR2. DDR1 was first identified in the Dictyostelium discoideum and was shown to mediate cell aggregation. DDR2 shares highly conserved sequences with DDR1. Both receptors are activated upon binding to collagen, one of the most abundant proteins in extracellular matrix. While DDR2 can only be activated by fibrillar collagen, particularly types I and III, DDR1 is mostly activated by type I and IV collagens. In contrast with classical growth factor tyrosine kinase receptors which display a rapid and transient activation, DDR1 and DDR2 are unique in that they exhibit delayed and sustained receptor phosphorylation upon binding to collagen. Recent studies have reported differential expression and mutations of DDR1 and DDR2 in several cancer types and indicate clearly that these receptors have to be taken into account as new players in the different aspects of tumor progression, from non-malignant to highly malignant and invasive stages. This review will discuss the current knowledge on the role of DDR1 and DDR2 in malignant transformation, cell proliferation, epithelial to mesenchymal transition, migratory, and invasive processes, and finally the modulation of the response to chemotherapy. These new insights suggest that DDR1 and DDR2 are new potential targets in cancer therapy.

Functional relationship between matrix metalloproteinase-11 and matrix metalloproteinase-14.

MMP-11 is a key factor in physiopathological tissue remodeling. As an active form is secreted, its activity must be tightly regulated to avoid detrimental effects. Although TIMP-1 and TIMP-2 reversibly inhibit MMP-11, another more drastic scenario, presumably via hydrolysis, could be hypothesized. In this context, we have investigated the possible implication of MMP-14, since it exhibits a spatiotemporal localization similar to MMP-11. Using native HFL1-produced MMP-11 and HT-1080-produced MMP-14 as well as recombinant proteins, we show that MMP-11 is a MMP-14 substrate. MMP-14 cleaves MMP-11 catalytic domain at the PGG(P1)-I(P1')LA and V/IQH(P1)-L(P1')YG scissile bonds, two new cleavage sites. Interestingly, a functional test showed a dramatical reduction in MMP-11 enzymatic activity when incubated with active MMP-14, whereas inactive point-mutated MMP-14 had no effect. This function is conserved between human and mouse. Thus, in addition to the canonical reversible TIMP-dependent inhibitory system, irreversible MMP proteolytic inactivation might occur by cleavage of the catalytic domain in a MMP-dependent manner. Since MMP-14 is produced by HT-1080 cancer cells, whereas MMP-11 is secreted by HFL1 stromal cells, our findings support the emerging importance of tumor-stroma interaction/cross-talk. Moreover, they highlight a Janus-faced MMP-14 function in the MMP cascade, favoring activation of several pro-MMPs, but limiting MMP-11 activity. Finally, both MMPs are active at the cell periphery. Since MMP-14 is present at the cell membrane, whereas MMP-11 is soluble into the cellular microenvironment, this MMP-14 function might represent one critical regulatory mechanism to control the extent of pericellular MMP-11 bioavailability and protect cells from excessive/inappropriate MMP-11 function.

[The tumoral stroma, a breeding ground for cancer cells]. / Le stroma tumoral - un terreau fertile pour la cellule cancéreuse.

Carcinomas are constituted by malignant epithelial cells and the tumor microenvironment also called tumoral stroma. The present non-exhaustive review will focus on cellular and molecular key events implicating cancer-associated fibroblasts (CAF) and the extracellular matrix (ECM) in the stroma remodeling processes occuring during tumor invasive steps. We will also discuss the place of the stroma in the cancerology today, the recent progresses done and its usefulness to design new therapies.

Adipocyte is a non-trivial, dynamic partner of breast cancer cells.

While the participation of adipocytes is well known in tissue architecture, energy supply and endocrine processes, their implication during natural cancer history is just beginning to unfold. An extensive review of the literature concerning the impact of resident adipocytes on breast cancer development/progression was performed. This review provides in vitro and in vivo evidence that adipocytes located close to invasive cancer cells, referred to as cancer-associated adipocytes (CAAs), are essential for breast tumor development/progression. Their deleterious function is dependent, at least partly, on their crosstalk with invasive cancer cells. Indeed, this event leads to dramatic phenotypic and/or functional modifications of both cell types. Adipocytes exhibit delipidation and acquire a fibroblast-like shape. In parallel, cancer cell aggressiveness is exacerbated through increased migratory and invasive properties. Moreover, obesity is currently a sign of poor prognosis in human carcinomas. In this context, a high number of "obese" resident adipocytes might be predicted to be detrimental. Accordingly, there are some similarities between the molecular alterations observed in hypertrophied adipocytes and in CAAs. How adipocytes function to favor tumorigenesis at the molecular level remains largely unknown. Nevertheless, progress has been made recently and molecular clues are starting to emerge. Deciphering the cellular and molecular mechanisms behind the adipocyte-cancer cell heterotypic crosstalk is of great interest since it might provide new targets for improving diagnosis/prognosis and for the design of innovative therapeutic strategies. They might also improve our understanding of the relationship between obesity/metabolic disorders and cancer risk and/or poor patient outcome.

Reduced secretion and expression of gelatinase profile in Toxoplasma gondii-infected human monocytic cells.

The apicomplexan Toxoplasma gondii, an obligate intracellular parasite, can infect humans and a wide range of vertebrates. Following oral infection, the parasite invades tissues by crossing non-permissive biological barriers such as the placenta or the blood-brain barrier. But the molecular mechanisms underlying migration of T. gondii remain poorly characterized. The crossing of various basal membranes and infiltration into the extracellular matrix by T. gondii could involve matrix metalloproteinases (MMPs). We demonstrated a decrease in proMMP-2 and proMMP-9 secretion by THP-1 cells at 24 and 48h post invasion with regulation at the mRNA level throughout infection. This down regulation was associated with a decrease in TIMP-2 secretion and an inhibition of its expression. Moreover, results showed an activation of MT1-MMP; its expression was regulated after 6, 24, and 48h.

Substitution of the unique cysteine residue of murine Hsp25 interferes with the protective activity of this stress protein through inhibition of dimer formation.

Murine small stress protein [heat shock protein 25 (Hsp25)] expression confers thermotolerance and protection against oxidative stress. Hsp25 is an oligomeric ATP-independent phospho-chaperone that can generate a glutathione-dependent pro-reducing state in cells that are normally devoid of small stress protein constitutive expression. Hsp25 contains only one cysteine residue (position 141) that is highly susceptible to oxidation. We have explored the significance of this reactive residue by generating a mutant in which cysteine-141 was substituted by an alanine residue (C141A mutant). We report here that the C141A mutant did not form dimers when expressed in either murine L929 or human HeLa cells, hence, demonstrating that cysteine-141 regulates Hsp25 dimer formation. The C141A mutant also interfered with the dimerization of human Hsp27, a constitutively expressed small stress protein in HeLa cells. The mutated polypeptide showed a decreased ability to multimerize, but its expression was still able to induce cellular protection against oxidative stress. The C141A mutant was, however, less efficient than the wild-type protein in counteracting staurosporine-induced apoptosis, and it showed no in vivo chaperone activity. Hence, the cellular protection mediated against different stressors may require specific structural organizations of Hsp25 that are differently altered by the mutation. Of interest, when expressed concomitantly with wild-type Hsp25, the C141A polypeptide induced a dominant-negative effect, a phenomenon that may result from the ability of small stress proteins to interact and multimerize with each other.