Human adipose-derived mesenchymal stem cells laden in gellan gum spongy-like hydrogels for volumetric muscle loss treatment.

BACKGROUND: volumetric muscle loss (VML) is a traumatic massive loss of muscular tissue which frequently leads to amputation, limb loss, or lifetime disability. The current medical intervention is limited to autologous tissue transfer, which usually leads to non-functional tissue recovery. Tissue engineering holds a huge promise for functional recovery. METHODS: in this work, we evaluated the potential of human adipose-derived mesenchymal stem cells (hASCs) pre-cultured in gellan gum based spongy-like hydrogels (SLHs). RESULTS: in vitro, hASCs were spreading, proliferating, and releasing growth factors and cytokines (i.e. fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor 1, interleukin-6 (IL-6), IL-8, IL-10, vascular endothelial growth factor) important for muscular regeneration. After implantation into a volumetric muscle loss (VML) mouse model, implants were degrading overtime, entirely integrating into the host between 4 and 8 weeks. In both SLH and SLH + hASCs defects, infiltrated cells were observed inside constructs associated with matrix deposition. Also, minimal collagen deposition was marginally observed around the constructs along both time-points. Neovascularization (CD31+vessels) and neoinnervation (ß-III tubulin+bundles) were significantly detected in the SLH + hASCs group, in relation to the SHAM (empty lesion). A higher density ofα-SA+and MYH7+cells were found in the injury site among all different experimental groups, at both time-points, in relation to the SHAM. The levels ofα-SA, MyoD1, and myosin heavy chain proteins were moderately increased in the SLH + hASCs group after 4 weeks, and in the hASCs group after 8 weeks, in relation to the SHAM. CONCLUSIONS: taken together, defects treated with hASCs-laden SLH promoted angiogenesis, neoinnervation, and the expression of myogenic proteins.

Selective Killing of BRCA2-Deficient Ovarian Cancer Cells via MRE11 Blockade.

The MRE11 nuclease is essential during DNA damage recognition, homologous recombination, and replication. BRCA2 plays important roles during homologous recombination and replication. Here, we show that effecting an MRE11 blockade using a prototypical inhibitor (Mirin) induces synthetic lethality (SL) in BRCA2-deficient ovarian cancer cells, HeLa cells, and 3D spheroids compared to BRCA2-proficient controls. Increased cytotoxicity was associated with double-strand break accumulation, S-phase cell cycle arrest, and increased apoptosis. An in silico analysis revealed Mirin docking onto the active site of MRE11. While Mirin sensitises DT40 MRE11+/- cells to the Top1 poison SN-38, it does not sensitise nuclease-dead MRE11 cells to this compound confirming that Mirin specifically inhibits Mre11 nuclease activity. MRE11 knockdown reduced cell viability in BRCA2-deficient PEO1 cells but not in BRCA2-proficient PEO4 cells. In a Mirin-resistant model, we show the downregulation of 53BP1 and DNA repair upregulation, leading to resistance, including in in vivo xenograft models. In a clinical cohort of human ovarian tumours, low levels of BRCA2 expression with high levels of MRE11 co-expression were linked with worse progression-free survival (PFS) (p = 0.005) and overall survival (OS) (p = 0.001). We conclude that MRE11 is an attractive SL target, and the pharmaceutical development of MRE11 inhibitors for precision oncology therapeutics may be of clinical benefit.

Unravelling the clinicopathological and functional significance of replication protein A (RPA) heterotrimeric complex in breast cancers.

Replication Protein A (RPA), a heterotrimeric complex consisting of RPA1, 2, and 3 subunits, is a single-stranded DNA (ssDNA)-binding protein that is critically involved in replication, checkpoint regulation and DNA repair. Here we have evaluated RPA in 776 pure ductal carcinomas in situ (DCIS), 239 DCIS that co-exist with invasive breast cancer (IBC), 50 normal breast tissue and 4221 IBC. Transcriptomic [METABRIC cohort (n = 1980)] and genomic [TCGA cohort (n = 1090)] evaluations were completed. Preclinically, RPA deficient cells were tested for cisplatin sensitivity and Olaparib induced synthetic lethality. Low RPA linked to aggressive DCIS, aggressive IBC, and shorter survival outcomes. At the transcriptomic level, low RPA tumours overexpress pseudogene/lncRNA as well as genes involved in chemical carcinogenesis, and drug metabolism. Low RPA remains linked with poor outcome. RPA deficient cells are sensitive to cisplatin and Olaparib induced synthetic lethality. We conclude that RPA directed precision oncology strategy is feasible in breast cancers.

Injectable laminin-biofunctionalized gellan gum hydrogels loaded with myoblasts for skeletal muscle regeneration.

Moderate muscular injuries that exceed muscular tissue's auto-healing capacity are still a topic of noteworthy concern. Tissue engineering appeared as a promising therapeutic strategy capable of overcoming this unmet clinical need. To attain such goal, herein we propose an in situ-crosslinking gellan gum (GG)-based hydrogel tethered with a skeletal muscle-inspired laminin-derived peptide RKRLQVQLSIRTC(Q) and encapsulated with skeletal muscle cells (SMCs). Pre-hydrogel solutions presented decreasing shear viscosity with increasing shear rate and shear stress, and required low forces for extrusion, validating their injectability. The GGDVS hydrogel was functionalized with Q-peptide with 30% of efficiency. C2C12 were able to adhere to the developed hydrogel, remained living and spreading 7 days post-encapsulation. Q-peptide release studies indicated that 25% of the unbound peptide can be released from the hydrogels up to 7 days, dependent on the hydrogel formulation. Treatment of a chemically-induced muscular lesion in mice with an injection of C2C12-laden hydrogels improved myogenesis, primarily promoted by the C2C12. In accordance, a high density of myoblasts (α-SA+ and MYH7+) were localized in tissues treated with the C2C12 (alone or encapsulated in the hydrogel). α-SA protein levels were significantly increased 8 weeks post-treatment with C2C12-laden hydrogels and MHC protein levels were increased in all experimental groups 4 weeks post-treatment, in relation to the SHAM. Neovascularization and neoinnervation was also detected in the defects. Altogether, this study indicates that C2C12-laden hydrogels hold great potential for skeletal muscle regeneration. STATEMENT OF SIGNIFICANCE: We developed an injectable gellan gum-based hydrogel for delivering C2C12 into localized myopathic model. The gellan gum was biofunctinalized with laminin-derived peptide to mimic the native muscular ECM. In addition, hydrogel was physically tuned to mimic the mechanical properties of native tissue. To the best of our knowledge, this formula was used for the first time under the context of skeletal muscle tissue regeneration. The injectability of the developed hydrogel provided non-invasive administration method, combined with a reliable microenvironment that can host C2C12 with nominal inflammation, indicated by the survival and adhesion of encapsulated cells post-injection. The treatment of skeletal muscle defect with the cell-laden hydrogel approach significantly enhanced the regeneration of localized muscular trauma.

Ligase 1 is a predictor of platinum resistance and its blockade is synthetically lethal in XRCC1 deficient epithelial ovarian cancers.

Rationale: The human ligases (LIG1, LIG3 and LIG4) are essential for the maintenance of genomic integrity by catalysing the formation of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxyl termini at single and double strand breaks in duplex DNA molecules generated either directly by DNA damage or during replication, recombination, and DNA repair. Whether LIG1, LIG3 and LIG4 can influence ovarian cancer pathogenesis and therapeutics is largely unknown. Methods: We investigated LIG1, LIG3 and LIG4 expression in clinical cohorts of epithelial ovarian cancers [protein level (n=525) and transcriptional level (n=1075)] and correlated to clinicopathological features and survival outcomes. Pre-clinically, platinum sensitivity was investigated in LIG1 depleted ovarian cancer cells. A small molecule inhibitor of LIG1 (L82) was tested for synthetic lethality application in XRCC1, BRCA2 or ATM deficient cancer cells. Results: LIG1 and LIG3 overexpression linked with aggressive phenotypes, platinum resistance and poor progression free survival (PFS). In contrast, LIG4 deficiency was associated with platinum resistance and worse PFS. In a multivariate analysis, LIG1 was independently associated with adverse outcome. In ovarian cancer cell lines, LIG1 depletion increased platinum cytotoxicity. L82 monotherapy was synthetically lethal in XRCC1 deficient ovarian cancer cells and 3D-spheroids. Increased cytotoxicity was linked with accumulation of DNA double strand breaks (DSBs), S-phase cell cycle arrest and increased apoptotic cells. L82 was also selectively toxic in BRCA2 deficient or ATM deficient cancer cells and 3D-spheroids. Conclusions: We provide evidence that LIG1 is an attractive target for personalization of ovarian cancer therapy.

FEN1 Blockade for Platinum Chemo-Sensitization and Synthetic Lethality in Epithelial Ovarian Cancers.

FEN1 plays critical roles in long patch base excision repair (LP-BER), Okazaki fragment maturation, and rescue of stalled replication forks. In a clinical cohort, FEN1 overexpression is associated with aggressive phenotype and poor progression-free survival after platinum chemotherapy. Pre-clinically, FEN1 is induced upon cisplatin treatment, and nuclear translocation of FEN1 is dependent on physical interaction with importin ß. FEN1 depletion, gene inactivation, or inhibition re-sensitizes platinum-resistant ovarian cancer cells to cisplatin. BRCA2 deficient cells exhibited synthetic lethality upon treatment with a FEN1 inhibitor. FEN1 inhibitor-resistant PEO1R cells were generated, and these reactivated BRCA2 and overexpressed the key repair proteins, POLß and XRCC1. FEN1i treatment was selectively toxic to POLß deficient but not XRCC1 deficient ovarian cancer cells. High throughput screening of 391,275 compounds identified several FEN1 inhibitor hits that are suitable for further drug development. We conclude that FEN1 is a valid target for ovarian cancer therapy.

Cell quality control mechanisms maintain stemness and differentiation potential of P19 embryonic carcinoma cells.

Given the relatively long life of stem cells (SCs), efficient mechanisms of quality control to balance cell survival and resistance to external and internal stress are required. Our objective was to test the relevance of cell quality control mechanisms for SCs maintenance, differentiation and resistance to cell death. We compared cell quality control in P19 stem cells (P19SCs) before and after differentiation (P19dCs). Differentiation of P19SCs resulted in alterations in parameters involved in cell survival and protein homeostasis, including the redox system, cardiolipin and lipid profiles, unfolded protein response, ubiquitin-proteasome and lysosomal systems, and signaling pathways controlling cell growth. In addition, P19SCs pluripotency was correlated with stronger antioxidant protection, modulation of apoptosis, and activation of macroautophagy, which all contributed to preserve SCs quality by increasing the threshold for cell death activation. Furthermore, our findings identify critical roles for the PI3K-AKT-MTOR pathway, as well as autophagic flux and apoptosis regulation in the maintenance of P19SCs pluripotency and differentiation potential.Abbreviations: 3-MA: 3-methyladenine; AKT/protein kinase B: thymoma viral proto-oncogene; AKT1: thymoma viral proto-oncogene 1; ATG: AuTophaGy-related; ATF6: activating transcription factor 6; BAX: BCL2-associated X protein; BBC3/PUMA: BCL2 binding component 3; BCL2: B cell leukemia/lymphoma 2; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CASP3: caspase 3; CASP8: caspase 8; CASP9: caspase 9; CL: cardiolipin; CTSB: cathepsin B; CTSD: cathepsin D; DDIT3/CHOP: DNA-damage inducible transcript 3; DNM1L/DRP1: dynamin 1-like; DRAM1: DNA-damage regulated autophagy modulator 1; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2S1/eIF2α: eukaryotic translation initiation factor 2, subunit alpha; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; ESCs: embryonic stem cells; KRT8/TROMA-1: cytokeratin 8; LAMP2A: lysosomal-associated membrane protein 2A; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NANOG: Nanog homeobox; NAO: 10-N-nonyl acridine orange; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; OPA1: OPA1, mitochondrial dynamin like GTPase; P19dCs: P19 differentiated cells; P19SCs: P19 stem cells; POU5F1/OCT4: POU domain, class 5, transcription factor 1; PtdIns3K: phosphatidylinositol 3-kinase; RA: retinoic acid; ROS: reactive oxygen species; RPS6KB1/p70S6K: ribosomal protein S6 kinase, polypeptide 1; SCs: stem cells; SOD: superoxide dismutase; SHC1-1/p66SHC: src homology 2 domain-containing transforming protein C1, 66 kDa isoform; SOX2: SRY (sex determining region Y)-box 2; SQSTM1/p62: sequestosome 1; SPTAN1/αII-spectrin: spectrin alpha, non-erythrocytic 1; TOMM20: translocase of outer mitochondrial membrane 20; TRP53/p53: transformation related protein 53; TUBB3/betaIII-tubulin: tubulin, beta 3 class III; UPR: unfolded protein response; UPS: ubiquitin-proteasome system.

PARP1 blockade is synthetically lethal in XRCC1 deficient sporadic epithelial ovarian cancers.

PARP1 inhibitor (Niraparib, Olaparib, Rucaparib) maintenance therapy improves progression-free survival in platinum sensitive sporadic epithelial ovarian cancers. However, biomarkers of response to PARPi therapy is yet to be clearly defined. XRCC1, a scaffolding protein, interacts with PARP1 during BER and SSBR. In a large clinical cohort of 525 sporadic ovarian cancers, high XRCC1 or high PARP1 protein levels was not only associated with aggressive phenotypes but was also significantly linked with poor progression-free survival (p = 0.048 & p = 0.001 respectively) and poor ovarian cancer-specific survival (p = 0.020 & p = 0.008 respectively). Pre-clinically, Olaparib and Talazoparib therapy were selectively toxic in XRCC1 deficient or knock-out platinum sensitive ovarian cancer cells in 2D and 3D models. Increased sensitivity was associated with DNA double-strand break accumulation, cell cycle arrest and apoptotic cell accumulation. We conclude that XRCC1 deficiency predicts sensitivity to PARP inhibitor therapy. PARP1 targeting is a promising new approach in XRCC1 deficient ovarian cancers.

ERCC1 Is a Predictor of Anthracycline Resistance and Taxane Sensitivity in Early Stage or Locally Advanced Breast Cancers.

Genomic instability could be a beneficial predictor for anthracycline or taxane chemotherapy. We interrogated 188 DNA repair genes in the METABRIC cohort (n = 1980) to identify genes that influence overall survival (OS). We then evaluated the clinicopathological significance of ERCC1 in early stage breast cancer (BC) (mRNA expression (n = 4640) and protein level, n = 1650 (test set), and n = 252 (validation)) and in locally advanced BC (LABC) (mRNA expression, test set (n = 2340) and validation (TOP clinical trial cohort, n = 120); and protein level (n = 120)). In the multivariate model, ERCC1 was independently associated with OS in the METABRIC cohort. In ER+ tumours, low ERCC1 transcript or protein level was associated with increased distant relapse risk (DRR). In ER-tumours, low ERCC1 transcript or protein level was linked to decreased DRR, especially in patients who received anthracycline chemotherapy. In LABC patients who received neoadjuvant anthracycline, low ERCC1 transcript was associated with higher pCR (pathological complete response) and decreased DRR. However, in patients with ER-tumours who received additional neoadjuvant taxane, high ERCC1 transcript was associated with a higher pCR and decreased DRR. High ERCC1 transcript was also linked to decreased DRR in ER+ LABC that received additional neoadjuvant taxane. ERCC1 based stratification is an attractive strategy for breast cancers.

ERCC1-XPF deficiency is a predictor of olaparib induced synthetic lethality and platinum sensitivity in epithelial ovarian cancers.

PURPOSE: PARP inhibitor maintenance therapy in platinum sensitive sporadic ovarian cancers improves progression free survival. However, biomarker for synthetic lethality in platinum sensitive sporadic disease is yet to be defined. ERCC1-XPF heterodimer is a key player in nucleotide excision repair (NER) involved in the repair of platinum induced DNA damage. In the current study, we tested whether ERCC1-XPF deficiency would predict synthetic lethality to the PARP inhibitor Olaparib and platinum sensitivity in ovarian cancers. METHODS: ERCC1, XPF and PARP1 protein expression was evaluated in tumors from a cohort of 331 patients treated at Nottingham University Hospitals and correlated to clinicopathological features and survival. Pre-clinically, ERCC1 and XPF was depleted in A2780 (platinum sensitive) and A2780cis (platinum resistant) ovarian cancer cell lines and tested for platinum sensitivity as well as for Olaparib induced synthetic lethality. RESULTS: Low ERCC1 was significantly associated with improved progression free survival (PFS) in patients with ovarian cancers in univariate (p = 0.001) and multivariate (p = 0.002) analysis. In addition, low ERCC1/low XPF (p = 0.003) or low ERCC1/low PARP1 (p = 0.0001) tumors was also linked to better PFS compared to high ERCC1/high XPF or high ERCC1/high PARP1 tumors. Pre-clinically, ERCC1 or XPF depletion not only increased platinum sensitivity but also increased toxicity to Olaparib therapy. Increased sensitivity was associated with DNA double strand breaks (DSBs) accumulation, cell cycle arrest and increased apoptosis. CONCLUSION: The data provide evidence that low ERCC1 is not only a predictor of platinum sensitivity but is also a promising biomarker for Olaparib induced synthetic lethality in ovarian cancers.

Targeting PARP1 in XRCC1-Deficient Sporadic Invasive Breast Cancer or Preinvasive Ductal Carcinoma In Situ Induces Synthetic Lethality and Chemoprevention.

: Targeting PARP1 for synthetic lethality is a new strategy for breast cancers harboring germline mutations in BRCA. However, these mutations are rare, and reactivation of BRCA-mediated pathways may result in eventual resistance to PARP1 inhibitor therapy. Alternative synthetic lethality approaches targeting more common sporadic breast cancers and preinvasive ductal carcinoma in situ (DCIS) are desirable. Here we show that downregulation of XRCC1, which interacts with PARP1 and coordinates base excision repair, is an early event in human breast cancer pathogenesis. XRCC1-deficient DCIS were aggressive and associated with increased risk of local recurrence. Human invasive breast cancers deficient in XRCC1 and expressing high PARP1 levels also manifested aggressive features and poor outcome. The PARP1 inhibitor olaparib was synthetically lethal in XRCC1-deficient DCIS and invasive breast cancer cells. We conclude that targeting PARP1 is an attractive strategy for synthetic lethality and chemoprevention in XRCC1-deficient breast cancers, including preinvasive DCIS. SIGNIFICANCE: These findings show that loss of XRCC1, which is associated with more malignant DCIS, can be exploited by PARP inhibition, suggesting its application as a promising therapeutic and chemoprevention strategy in XRCC1-deficient tumor cells.

Mitochondrial biology in cancer stem cells.

Cancer stem cells (CSCs) have been suggested to be responsible for tumor re-growth and relapse. Physiological and morphological knowledge of CSCs may be essential for the development of new therapeutic strategies targeting cancer development, progression, and recurrence. Current research is focused on a deeper understanding of CSCs metabolic profiles, taking into consideration their energy demands. Energy metabolism and mitochondrial function are important factors operating on stemness maintenance and cell fate specification. Due to the role of mitochondria as central hubs in the overall cell metabolism and death and survival pathways, research on their physiology in CSCs is of paramount importance to decipher mechanisms underlying their therapy-resistant phenotype. In this review, we focus on CSCs mitochondrial biology and mitochondria-related signaling pathways that contribute to CSCs survival and maintenance, thereby representing possible therapeutic targets.

Melatonin antiproliferative effects require active mitochondrial function in embryonal carcinoma cells.

Although melatonin oncostatic and cytotoxic effects have been described in different types of cancer cells, the specific mechanisms leading to its antitumoral effects and their metabolic context specificity are still not completely understood. Here, we evaluated the effects of melatonin in P19 embryonal carcinoma stem cells (CSCs) and in their differentiated counterparts, cultured in either high glucose medium or in a galactose (glucose-free) medium which leads to glycolytic suppression and increased mitochondrial metabolism. We found that highly glycolytic P19 CSCs were less susceptible to melatonin antitumoral effects while cell populations relying on oxidative metabolism for ATP production were more affected. The observed antiproliferative action of melatonin was associated with an arrest at S-phase, decreased oxygen consumption, down-regulation of BCL-2 expression and an increase in oxidative stress culminating with caspase-3-independent cell death. Interestingly, the combined treatment of melatonin and dichloroacetate had a synergistic effect in cells grown in the galactose medium and resulted in an inhibitory effect in the highly resistant P19 CSCs. Melatonin appears to exert its antiproliferative activity in P19 carcinoma cells through a mitochondrially-mediated action which in turn allows the amplification of the effects of dichloroacetate, even in cells with a more glycolytic phenotype.

Mitochondria in cancer stem cells: a target for therapy.

Complete knowledge about the evolution of the carcinogenic process has to include cancer stem cells (CSCs), which are essential to understand tumor occurrence, recurrence, and also its reduction rate after radio- and/or chemotherapeutic treatments. Understanding CSCs physiology and metabolism may be crucial for the development of novel effective therapies. Therefore, being mitochondria an undeniable target for cancer therapy and a central hub in metabolism and cell and death decisions, it is essential to take this organelle into account and explore its actions and involvements in the context of CSCs physiology. In this review, we focus on recent patents and discoveries about mitochondrial bioenergetics and physiology of CSCs. A full understanding of the role of mitochondrial activity in CSCs and the creation of new strategies, methods and discoveries to support actual treatments with novel ones are of pivotal importance in order to ultimately eradicate cancer.

In vitro surfactant structure-toxicity relationships: implications for surfactant use in sexually transmitted infection prophylaxis and contraception.

BACKGROUND: The need for woman-controlled, cheap, safe, effective, easy-to-use and easy-to-store topical applications for prophylaxis against sexually transmitted infections (STIs) makes surfactant-containing formulations an interesting option that requires a more fundamental knowledge concerning surfactant toxicology and structure-activity relationships. METHODOLOGY/PRINCIPAL FINDINGS: We report in vitro effects of surfactant concentration, exposure time and structure on the viability of mammalian cell types typically encountered in the vagina, namely, fully polarized and confluent epithelial cells, confluent but non-polarized epithelial-like cells, dendritic cells, and human sperm. Representatives of the different families of commercially available surfactants--nonionic (Triton X-100 and monolaurin), zwitterionic (DDPS), anionic (SDS), and cationic (C(n)TAB (n = 10 to 16), C(12)PB, and C(12)BZK)--were examined. Triton X-100, monolaurin, DDPS and SDS were toxic to all cell types at concentrations around their critical micelle concentration (CMC) suggesting a non-selective mode of action involving cell membrane destabilization and/or destruction. All cationic surfactants were toxic at concentrations far below their CMC and showed significant differences in their toxicity toward polarized as compared with non-polarized cells. Their toxicity was also dependent on the chemical nature of the polar head group. Our results suggest an intracellular locus of action for cationic surfactants and show that their structure-activity relationships could be profitably exploited for STI prophylaxis in vaginal gel formulations. The therapeutic indices comparing polarized epithelial cell toxicity to sperm toxicity for all surfactants examined, except C(12)PB and C(12)BZK, does not justify their use as contraceptive agents. C(12)PB and C(12)BZK are shown to have a narrow therapeutic index recommending caution in their use in contraceptive formulations. CONCLUSIONS/SIGNIFICANCE: Our results contribute to understanding the mechanisms involved in surfactant toxicity, have a predictive value with regard to their safety, and may be used to design more effective and less harmful surfactants for use in topical applications for STI prophylaxis.