Genetic Polymorphism of ITGA2 C807T Collagen Receptor Encoding Gene of Aspirin Therapy among Javanese-Indonesian Healthy Respondents

BACKGROUND: Aspirin is an antiplatelet drug commonly administered as primary and secondary prophylaxis to prevent thromboembolic events. However, there has been a common incidence of aspirin resistance that leads to a recurrent cerebrovascular disease. One of the causes of such event is the genetic polymorphisms of the integrin alpha-2 (ITGA2) gene that encodes the glycoprotein Ia (GPIa) receptor in the pharmacodynamics of aspirin. AIM: This study analyzed the genetic polymorphism of ITGA2 as the GPIa collagen receptor encoding gene of aspirin therapy among healthy Javanese, the largest ethnic group in Indonesia. METHODS: This cross-sectional study involved 100 respondents who met the inclusion criteria with their blood sample taken for DNA isolation. Identification of genetic polymorphism in the target SNPs was done using the PCR-RFLP method with 5’-CCTTAAAGCTACCGGCCCATGT-3’ forward primer and 5’-TTGGCCTATTAGCACCAAAACTTACC-3’ reverse primer as well as Hpy188Irestriction enzyme to fragment the target at position 244 in the C base. RESULTS: This study found that the dominant genotype and allele were CT (51%) and C (66.5%), respectively. CONCLUSION: The allele frequency of ITGA2 gene in this study was similar to that of the populations in other Asian countries. Further research regarding the effects of ITGA2 C807T polymorphism on the pharmacodynamics of aspirin as an antiplatelet is recommended to minimize atherothrombotic events and examine its interactions as a biomarker of the risk and prognosis of some cancer types.

The Collagen Receptor uPARAP in Malignant Mesothelioma: A Potential Diagnostic Marker and Therapeutic Target

Malignant mesothelioma (MM) is a highly aggressive cancer with limited therapeutic options. We have previously shown that the endocytic collagen receptor, uPARAP, is upregulated in certain cancers and can be therapeutically targeted. Public RNA expression data display uPARAP overexpression in MM. Thus, to evaluate its potential use in diagnostics and therapy, we quantified uPARAP expression by immunohistochemical H-score in formalin-fixed paraffin-embedded bioptic/surgical human tissue samples and tissue microarrays. We detected pronounced upregulation of uPARAP in the three main MM subtypes compared to non-malignant reactive mesothelial proliferations, with higher expression in sarcomatoid and biphasic than in epithelioid MM. The upregulation appeared to be independent of patients’ asbestos exposure and unaffected after chemotherapy. Using immunoblotting, we demonstrated high expression of uPARAP in MM cell lines and no expression in a non-malignant mesothelial cell line. Moreover, we showed the specific internalization of an anti-uPARAP monoclonal antibody by the MM cell lines using flow cytometry-based assays and confocal microscopy. Finally, we demonstrated the sensitivity of these cells towards sub-nanomolar concentrations of an antibody-drug conjugate formed with the uPARAP-directed antibody and a potent cytotoxin that led to efficient, uPARAP-specific eradication of the MM cells. Further studies on patient cohorts and functional preclinical models will fully reveal whether uPARAP could be exploited in diagnostics and therapeutic targeting of MM.

DDR1 Affects Metabolic Reprogramming in Breast Cancer Cells by Cross-Talking to the Insulin/IGF System

The insulin receptor isoform A (IR-A), a dual receptor for insulin and IGF2, plays a role in breast cancer (BC) progression and metabolic reprogramming. Notably, discoidin domain receptor 1 (DDR1), a collagen receptor often dysregulated in cancer, is involved in a functional crosstalk and feed forward loop with both the IR-A and the insulin like growth factor receptor 1 (IGF1R). Here, we aimed at investigating whether DDR1 might affect BC cell metabolism by modulating the IGF1R and/or the IR. To this aim, we generated MCF7 BC cells engineered to stably overexpress either IGF2 (MCF7/IGF2) or the IR-A (MCF7/IR-A). In both cell models, we observed that DDR1 silencing induced a significant decrease of total ATP production, particularly affecting the rate of mitochondrial ATP production. We also observed the downregulation of key molecules implicated in both glycolysis and oxidative phosphorylation. These metabolic changes were not modulated by DDR1 binding to collagen and occurred in part in the absence of IR/IGF1R phosphorylation. DDR1 silencing was ineffective in MCF7 knocked out for DDR1. Taken together, these results indicate that DDR1, acting in part independently of IR / IGF1R stimulation, might work as a novel regulator of BC metabolism and should be considered as putative target for therapy in BC.

CRISPR-edited megakaryocytes for rapid screening of platelet gene functions

Human anucleate platelets cannot be directly modified using traditional genetic approaches. Instead, studies of platelet gene function depend on alternative models. Megakaryocytes (the nucleated precursor to platelets) are the nearest cell to platelets in origin, structure, and function. However, achieving consistent genetic modifications in primary megakaryocytes has been challenging, and the functional effects of induced gene deletions on human megakaryocytes for even well-characterized platelet genes (eg, ITGA2B) are unknown. Here we present a rapid and systematic approach to screen genes for platelet functions in CD34+ cell-derived megakaryocytes called CRIMSON (CRISPR-edited megakaryocytes for rapid screening of platelet gene functions). By using CRISPR/Cas9, we achieved efficient nonviral gene editing of a panel of platelet genes in megakaryocytes without compromising megakaryopoiesis. Gene editing induced loss of protein in up to 95% of cells for platelet function genes GP6, RASGRP2, and ITGA2B; for the immune receptor component B2M; and for COMMD7, which was previously associated with cardiovascular disease and platelet function. Gene deletions affected several select responses to platelet agonists in megakaryocytes in a manner largely consistent with those expected for platelets. Deletion of B2M did not significantly affect platelet-like responses, whereas deletion of ITGA2B abolished agonist-induced integrin activation and spreading on fibrinogen without affecting the translocation of P-selectin. Deletion of GP6 abrogated responses to collagen receptor agonists but not thrombin. Deletion of RASGRP2 impaired functional responses to adenosine 5′-diphosphate (ADP), thrombin, and collagen receptor agonists. Deletion of COMMD7 significantly impaired multiple responses to platelet agonists. Together, our data recommend CRIMSON for rapid evaluation of platelet gene phenotype associations.

Role of Discoidin Domain Receptor 2 in Craniofacial Bone Regeneration

Bone loss caused by trauma, neoplasia, congenital defects, or periodontal disease is a major cause of disability and human suffering. Skeletal progenitor cell–extracellular matrix interactions are critical for bone regeneration. Discoidin domain receptor 2 (DDR2), an understudied collagen receptor, plays an important role in skeletal development. Ddr2 loss-of-function mutations in humans and mice cause severe craniofacial and skeletal defects, including altered cranial shape, dwarfing, reduced trabecular and cortical bone, alveolar bone/periodontal defects, and altered dentition. However, the role of this collagen receptor in craniofacial regeneration has not been examined. To address this, calvarial subcritical-size defects were generated in wild-type (WT) and Ddr2-deficient mice. The complete bridging seen in WT controls at 4 wk postsurgery was not observed in Ddr2-deficient mice even after 12 wk. Quantitation of defect bone area by micro–computed tomography also revealed a 50% reduction in new bone volume in Ddr2-deficient mice. Ddr2 expression during calvarial bone regeneration was measured using Ddr2-LacZ knock-in mice. Expression was restricted to periosteal surfaces of uninjured calvarial bone and, after injury, was detected in select regions of the defect site by 3 d postsurgery and expanded during the healing process. The impaired bone healing associated with Ddr2 deficiency may be related to reduced osteoprogenitor or osteoblast cell proliferation and differentiation since knockdown/knockout of Ddr2 in a mesenchymal cell line and primary calvarial osteoblast cultures reduced osteoblast differentiation while Ddr2 overexpression was stimulatory. In conclusion, Ddr2 is required for cranial bone regeneration and may be a novel target for therapy.