Biochemical and morpho-mechanical properties, and structural organization of rat tail tendon collagen in diet-induced obesity model.

We have investigated the impact of obesity on the structural organization, morpho-mechanical properties of collagen fibers from rat tail tendon fascicles (RTTFs). Polarized Raman microspectroscopy showed that the collagen bands 855, 875, 938, and 960 cm-1 as well as those 1631 and 1660 cm-1 were affected by diet. Mechanical properties exhibited an increase in the yield strength from control (CTRL) to high fat (HF) diet (9.60 ± 1.71 and 13.09 ± 1.81 MPa) (p < 0.01) and ultimate tensile strength (13.12 ± 2.37 and 18.32 ± 2.83 MPa) (p < 0.05) with no significant change in the Young's Modulus. During mechanical, the band at 875 cm-1 exhibited the most relevant frequency shift (2 cm-1). The intensity of those at 855, 875, and 938 cm-1 in HF collagen displayed a comparable response to mechanical stress as compared to CTRL collagen with no significant diet-related changes in the Full Width at Half Maximum. Second harmonic generation technique revealed i) similar fiber straightness (0.963 ± 0.004 and 0.965 ± 0.003) and ii) significant changes in fibers diameter (1.48 ± 0.07 and 1.52 ± 0.08 µm) (p < 0.05) and length (22.06 ± 2.38 and 29.00 ± 3.76 µm) (p < 0.001) between CTRL and HF diet, respectively. The quantification of advanced glycation end products (AGEs) revealed an increase in both carboxymethyl-lysine and total fluorescence AGEs from CTRL to HF RTTFs.

BRAF V600-Mutated Metastatic Melanoma and Targeted Therapy Resistance: An Update of the Current Knowledge.

Melanoma is the most common cause of death in skin cancer due to its high metastatic potential. While targeted therapies have improved the care of patients with metastatic melanoma harboring the BRAFV600E mutation, these treatments are associated with a high frequency of resistance. Resistance factors are related to cellular adaptation as well as to changes in the tumor microenvironment. At the cellular level, resistance involves mutations, overexpression, activation, or inhibition of effectors involved in cell signaling pathways such as MAPK, PI3K/AKT, MITF, and epigenetic factors (miRNAs). In addition, several components of the melanoma microenvironment, such as soluble factors, collagen, and stromal cells also play a crucial role in this resistance. In fact, extracellular matrix remodeling impacts the physical and chemical properties with changes in the stiffness and acidity, respectively of the microenvironment. The cellular and immune components of the stroma are also affected, including immune cells and CAF. The aim of this manuscript is to review the mechanisms responsible for resistance to targeted therapies in BRAFV600E-mutated metastatic melanoma.

An integrated approach to investigate age-related modifications of morphological, mechanical and structural properties of type I collagen.

The main propose of this study is to characterize the impact of chronological aging on mechanical, structural, biochemical, and morphological properties of type I collagen. We have developed an original approach combining a stress-strain measurement device with a portable Raman spectrometer to enable simultaneous measurement of Raman spectra during stress vs strain responses of young adult, adult and old rat tail tendon fascicles (RTTFs). Our data showed an increase in all mechanical properties such as Young's modulus, yield strength, and ultimate tensile strength with aging. At the molecular level, Raman data revealed that the most relevant frequency shift was observed at 938 cm-1 in Old RTTFs, which is assigned to the C-C. This suggested a long axis deformation of the peptide chains in Old RTTFs during tensile stress. In addition, the intensity of the band at 872 cm-1, corresponding to hydroxyproline decreased for young adult RTTFs and increased for the adult ones, while it remained unchanged for Old RTTFs during tensile stress. The amide III band (1242 and 1265 cm-1) as well as the band ratios I1631/ I1663 and I1645 / I1663 responses to tensile stress were depending on mechanical phases (toe, elastic and plastic). The quantification of advanced glycation end-products by LC-MS/MS and spectrofluorometry showed an increase in their content with aging. This suggested that the accumulation of such products was correlated to the alterations observed in the mechanical and molecular properties of RTTFs. Analysis of the morphological properties of RTTFs by SHG combined with CT-FIRE software revealed an increase in length and straightness of collagen fibers, whereas their width and wavy fraction decreased. Our integrated study model could be useful to provide additional translational information to monitor progression of diseases related to collagen remodeling in musculoskeletal disorders. STATEMENT OF SIGNIFICANCE: Type I collagen is the major component of the extracellular matrix. Its architectural and structural organization plays an important role in the mechanical properties of many tissues at the physiological and pathological levels. The objective of this work is to develop an integrated approach to bring a new insight on the impact of chronological aging on the structural organization and mechanical properties of type I collagen. We combined a portable Raman spectrometer with a mechanical tensile testing device in order to monitor in real time the changes in the Raman fingerprint of type I collagen fibers during the mechanical stress. Raman spectroscopy allowed the identification of the type I collagen bonds that were affected by mechanical stress in a differential manner with aging.

Degradation of p53 by HPV16-E6 variants isolated from cervical cancer specimens of Moroccan women.

Cervical cancer is the second most diagnosed cancer in Moroccan women. The main etiological factor for developing cervical cancer is the persistent infection with HPV16. Genetic studies have reported the occurrence of amino acid variations within the E6 oncoprotein that promotes host cell transformation by targeting p53 for degradation. To verify the biological effects of E6 polymorphisms towards p53 degradation, HPV16-E6 prototype and 7 variants isolated from cervical cancer biopsies of Moroccan women were evaluated for their activities by transient expression assays using pcDNA3.1-E6 constructs in C33A cell line. Expression of E6 genes in transfected cells was detected with reverse transcription PCR (RT-PCR), then, p53 levels were evaluated by western blot analysis. Significant dissimilarities in p53 degradation activities of HPV16-E6 prototype and intratypic variants were noticed. As compared to the prototype, the highest p53 degradation were exhibited by the African variants Af2-a/r, Af1-d/G295 and Af2-a/G285 (p < 0.001), followed by the European variants E- C442/G350 and E-G350/r (p < 0.01), then, the North American variant NA1-b/r (p < 0.05). The inter-variant differences were statistically significant between Af2-a/r variant and the North American variants NA1-b/r and NA1 (p < 0.05). Thus, the Af2-a/r variant was significantly more active in degrading p53 in our in vitro experiments (p < 0.0001). Our findings support the fact that HPV16-E6 variations have a biological impact on degrading p53, and so, represent a significant carcinogenic potential for developing cervical cancer.

Collagen and Discoidin Domain Receptor 1 Partnership: A Multifaceted Role in the Regulation of Breast Carcinoma Cell Phenotype.

Type I collagen, the major components of breast interstitial stroma, is able to regulate breast carcinoma cell behavior. Discoidin domain receptor 1 (DDR1) is a type I collagen receptor playing a key role in this process. In fact, collagen/DDR1 axis is able to trigger the downregulation of cell proliferation and the activation of BIK-mediated apoptosis pathway. The aim of this review is to discuss the role of two important factors that regulate these processes. The first factor is the level of DDR1 expression. DDR1 is highly expressed in epithelial-like breast carcinoma cells, but poorly in basal-like ones. Moreover, DDR1 undergoes cleavage by MT1-MMP, which is highly expressed in basal-like breast carcinoma cells. The second factor is type I collagen remodeling since DDR1 activation depends on its fibrillar organization. Collagen remodeling is involved in the regulation of cell proliferation and apoptosis through age- and proteolysis-related modifications.

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 changes in molecular organization of type I collagen in tendon as probed by polarized SHG and Raman microspectroscopy.

Type I Collagen is one of the most abundant proteins of the extracellular matrix of the most organs. During chronological aging or in diseases, type I collagen undergoes biochemical and structural changes which can impact biomechanical and physiological properties of organs. In this study, we have investigated the age-related changes in the molecular organization of type I collagen in rat tails tendon using polarized Raman spectroscopy. Our results show that Amide I, amide III as well as the bands related to proline and hydroxyproline are highly sensitive to polarization and age-related. On the other hand, 1453 and 1270 cm-1 do not show any preferential orientation. Depolarization and anisotropic ratios were used to provide information about the changes in orientation of collagen fibers with aging. The anisotropy degree of Raman bands increase from adult to old collagen, indicating a higher collagen fibers alignment to the fascicle backbone axis in old tendons, and consequently a higher straightness of collagen fibers. These data were correlated to those obtained using polarized second harmonic generation technique. Polarized Raman mapping showed a more homogeneous spatial distribution of collagen fibers alignment to the fascicle axis in old tendon. This confirms a higher straightness of collagen fiber with aging.

Collagen type 1 promotes survival of human breast cancer cells by overexpressing Kv10.1 potassium and Orai1 calcium channels through DDR1-dependent pathway.

Collagen type 1 is among the tumor microenvironment (TM) factors, that regulates proliferation, survival, migration and invasion. Ion channels are key players in interactions between tumor cells and TM. Kv10.1 has been shown to play an essential role in breast cancer cell proliferation and migration by permitting Ca2+ influx notably via Orai1. Here, we show that human breast cancer (BC) cells growing, in culture media completely devoid of the serum and seeded on collagen 1 coating, exhibited less apoptotic rate and a decrease in Bax expression when compared to those grown on plastic. The survival conferred by collagen 1 was completely abolished by removing extracellular Ca2+ from the culture medium. In addition, Ca2+ entry was increased in collagen 1 condition along with increased Kv10.1 and Orai1 expressions. Moreover, collagen 1 was able to increase co-localization of Kv10.1 and Orai1 on the plasma membrane. Interestingly, silencing of Kv10.1 and Orai1 reduced survival and Ca2+influx without any additive effect. This calcium-dependent survival is accompanied by the activation of ERK1/2, and its pharmacological inhibition completely abolished the increase in Kv10.1 and Orai1 expressions, activities, and the cell survival induced by collagen 1. Moreover, both Kv10.1 and Orai1 knockdown reduced ERK1/2 activation but not Akt. Finally, DDR1 silencing but not ß1-integrin reduced the collagen induced survival, ERK1/2 phosphorylation and the expression of Kv10.1 and Orai1. Together these data show that the Kv10.1/Orai1 complex is involved in BC cell survival and this is dependent on collagen 1/DDR1 pathway. Therefore, they represent a checkpoint of tumor progression induced by the tumor microenvironment.

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.].

Biological study of the effect of water soluble [N-(2-hydroxybenzyl)-L-aspartato] gallium complexes on breast carcinoma and fibrosarcoma cells.

Two water soluble gallium complexes described as [Ga(III)LCl], where L is the deprotonated form of N-2-hydroxybenzyl aspartic acid derivatives, were synthesized and characterized by (1)H NMR, (13)C NMR, FT-IR, mass spectrometry, and elemental analysis. The 2-(5-chloro-2-hydroxybenzylamino)succinic acid derivative (GS2) has been found to be a promising anticancer drug candidate. This compound was found to be more cytotoxic against human breast carcinoma MDA-MB231 and fibrosarcoma HT-1080 cell lines than the unsubstituted derivative and GaCl3. GS2 was able to induce apoptosis through downregulation of AKT phosphorylation, G2M arrest in cell cycle, and caspase 3/7 pathway. This gallium complex was found to induce an increase in mitochondrial ROS level in HT-1080 cells but not in MDA-MB231 cells. This suggests that the mechanism of action of GS2 would not be mediated by the drug-induced oxidative stress but probably by directly and indirectly inhibiting the AKT cell-signaling pathway.

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.