Sex: Male
Education:
- Master of Philosophy in Bioscience Enterprise, University of Cambridge, 2006
- Doctor of Philosophy in Biochemistry and Molecular Biology, The Australian National University, 1994
Field of Specialization
Biotechnology
Genetics
Researches:
Article title: CircPVT1 attenuates negative regulation of NRAS by let-7 and drives cancer cells towards oncogenicity
Authors: Joshua Miguel C. Danac and Reynaldo L. Garcia
Publication title: Scientific Reports 11(9021), April 2021
Abstract:
Circular RNAs have emerged as functional regulatory molecules whose aberrant expression has been linked to diverse pathophysiological processes. Here, we report that circPVT1 interferes with let-7 binding to NRAS, confirming this axis as one route by which circPVT1 can instigate an oncogenic program in A549 lung cancer cells and HCT116 colorectal cancer cells. CircPVT1 knockdown significantly reduced NRAS levels and attenuated cancer hallmark phenotypes such as proliferation, migration, resistance to apoptosis, cytoskeletal disorganization, and epithelial-mesenchymal transition. The effects of circPVT1 knockdown were at least partially rescued by blocking binding of let-7 to NRAS 3′UTR with a target protector, suggesting that a circPVT1/let-7/NRAS axis exists and acts in cells to reverse NRAS downregulation and favor oncogenicity. While the phenotypic effects of circPVT1 knockdown may be attributable to the global action of circPVT1, the target protection assays resolved the relative contribution of the circPVT1/let-7/NRAS axis specifically.
Full text link https://tinyurl.com/hmzvxd3t
Article title: Exosomal microRNAs in colorectal cancer: Overcoming barriers of the metastatic cascade (Review)
Authors: Joshua Miguel C Danac, Aileen Geobee G Uy, Reynaldo L Garcia
Publication title: International Journal of Molecular Medicine 47(6):112, April 2021
Abstract:
The journey of cancer cells from a primary tumor to distant sites is a multi-step process that involves cellular reprogramming, the breaking or breaching of physical barriers and the preparation of a pre-metastatic niche for colonization. The loss of adhesion between cells, cytoskeletal remodeling, the reduction in size and change in cell shape, the destruction of the extracellular matrix, and the modification of the tumor microenvironment facilitate migration and invasion into surrounding tissues. The promotion of vascular leakiness enables intra-and extravasation, while angiogenesis and immune suppression help metastasizing cells become established in the new site. Tumor-derived exosomes have long been known to harbor microRNAs (miRNAs or miRs) that help prepare secondary sites for metastasis; however, their roles in the early and intermediate steps of the metastatic cascade are only beginning to be characterized. The present review article presents a summary and discussion of the miRNAs that form part of colorectal cancer (CRC)-derived exosomal cargoes and which play distinct roles in epithelial to mesenchymal plasticity and metastatic organotropism. First, an overview of epithelial-to-mesenchymal transition (EMT), metastatic organotropism, as well as exosome biogenesis, cargo sorting and uptake by recipient cells is presented. Lastly, the potential of these exosomal miRNAs as prognostic biomarkers for metastatic CRC, and the blocking of these as a possible therapeutic intervention is discussed.
Full text link https://tinyurl.com/47a3myey
Article title: Mutation-Associated Phenotypic Heterogeneity in Novel and Canonical PIK3CA Helical and Kinase Domain Mutants
Authors: Arman Ali Ghodsinia J-Ann Marie T. Lego and Reynaldo L. Garcia
Publication title: Cells 9(5), April 2020
Abstract:
Phosphatidylinositol 3-kinase, catalytic subunit alpha (PIK3CA) is an oncogene often mutated in colorectal cancer (CRC). The contribution of PIK3CA mutations in acquired resistance to anti-epidermal growth factor receptor (EGFR) therapy is well documented, but their prognostic and predictive value remain unclear. Domain-and exon-specific mutations are implicated in either favorable or poor prognoses, but there is paucity in the number of mutations characterized outside of the mutational hotspots. Here, two novel non-hotspot mutants-Q661K in exon 13 and C901R in exon 19-were characterized alongside the canonical exon 9 E545K and exon 20 H1047R mutants in NIH3T3 and HCT116 cells. Q661K and E545K both map to the helical domain, whereas C901R and H1047R map to the kinase domain. Results showed variable effects of Q661K and C901R on morphology, cellular proliferation, apoptosis resistance, and cytoskeletal reorganization, with both not having any effect on cellular migration. In comparison, E545K markedly promoted proliferation, survival, cytoskeletal reorganization, migration, and spheroid formation, whereas H1047R only enhanced the first three. In silico docking suggested these mutations negatively affect binding of the p85 alpha regulatory subunit to PIK3CA, thereby relieving PIK3CA inhibition. Altogether, these findings support intra-domain and mutation-specific variability in oncogenic readouts, with implications in degree of aggressiveness.
Full text link https://tinyurl.com/arwpr2wx
Article title: Identification and validation of mRNA 3'untranslated regions of DNMT3B and TET3 as novel competing endogenous RNAs of the tumor suppressor PTEN
Authors: Kenneth Anthony R. Roquid, Krizelle Mae M. Alcantara, Reynaldo L. Garcia
Publication title: International Journal of Oncology 56(2):544-558, February 2020
Abstract:
PTEN inactivation is a frequent event in oncogenesis. Multiple regulatory mechanisms such as promoter hypermethylation, antisense regulation, histone modifications, targeting by microRNAs (miRNAs/miRs) and regulation by transcription factors have all been shown to affect the tumor suppressor functions of PTEN. More recently, the functional involvement of competing endogenous RNAs (ceRNAs) in miRNA‑dependent and coding‑independent regulation of genes shed light on the highly nuanced control of PTEN expression. The present study has identified and validated DNA methyltransferase 3β (DNMT3B) and TET methylcytosine dioxygenase 3 (TET3) as novel ceRNAs of PTEN, with which they share multiple miRNAs, in HCT116 colorectal cancer cells. miR‑4465 was identified and characterized as a miRNA that directly targets and regulates all 3 transcripts via their 3'untranslated regions (3'UTRs) through a combination of luciferase reporter assays, abrogation of miRNA response elements (MREs) via site‑directed mutagenesis, target protection of MREs with locked nucleic acids, RT‑qPCR assays and western blot analysis. Competitive miRNA sequestration was demonstrated upon reciprocal 3'UTR overexpression and siRNA‑mediated knockdown of their respective transcripts. Overexpression of DNMT3B or TET3 3'UTR promoted apoptosis and decreased migratory capacity, potentially because of shared miRNA sequestration and subsequent activation of PTEN expression. Knockdown of TET3 and DNMT3B decoupled their protein‑coding from miRNA‑dependent, coding‑independent functions. Furthermore, the findings suggested that the phenotypic outcome of ceRNAs is dictated largely by the number of shared miRNAs, and predictably, by the existence of other ceRNA networks in which they participate. Taken together, the findings of the present study identified DNMT3B and TET3 as novel ceRNAs of PTEN that may impact its dose‑sensitive tumor suppressive function.
Full text link https://tinyurl.com/2tpjfzbs
Article title: Non-Redundant and Overlapping Oncogenic Readouts of Non-Canonical and Novel Colorectal Cancer KRAS and NRAS Mutants
Authors: Krizelle Mae M. Alcantara, Joshua Reginald P. Malapit , Ryan Timothy D. Yu, Jose Antonio Ma. G. Garrido, et al.
Publication title: Cells 8(12), December 2019
Abstract:
RAS oncogene family members are molecular switches of signaling pathways that control cell growth, proliferation, differentiation, and survival. In colorectal cancer, Kirsten-RAS (KRAS) and neuroblastoma-RAS (NRAS) are the commonly mutated isoforms. Activating mutations in RAS result in cellular transformation independent of upregulated epidermal growth factor receptor (EGFR)-initiated signaling. The present study characterized the functional consequences of non-canonical/novel KRAS and NRAS mutants identified in a targeted next-generation sequencing study of colorectal cancer specimens from Filipino patients. In vitro assays in NIH3T3 cells showed that similar to the canonical KRAS G12D mutant, overexpression of KRAS G12S, A59T, and Y137C, but not NRAS G12D and NRAS A11V, confer higher proliferation and migration rates. HCT116 cells transfected with the novel NRAS A11V and the canonical NRAS G12D, but not the KRAS mutants, display enhanced resistance to apoptosis. All four non-canonical/novel KRAS and NRAS mutants induce gross changes in F-actin cytoskeletal organization and cellular morphology of NIH3T3 cells. Only KRAS G12S and KRAS A59T appear to deregulate extracellular signal-regulated kinase (ERK) and its downstream target ETS transcription factor ELK1 (ELK1). Elucidation of differential effector engagement responsible for the variable phenotypic readouts of the mutants is warranted. If validated by mouse studies and clinical correlates, these can have wider implications in choosing treatment options.
Full text link https://tinyurl.com/y83jka8h
Article title: MicroRNA‑92a promotes cell proliferation, migration and survival by directly targeting the tumor suppressor gene NF2 in colorectal and lung cancer cells
Authors: Krizelle Mae M. Alcantara and Reynaldo L. Garcia
Publication title: Oncology Reports 41(4), February 2019
Abstract:
Inactivation of the tumor suppressor protein Merlin leads to the development of benign nervous system tumors in neurofibromatosis type2 (NF2). Documented causes of Merlin inactivation include deleterious mutations in the encoding neurofibromin2 gene (NF2) and aberrant Merlin phosphorylation leading to proteasomal degradation. Rare somatic NF2 mutations have also been detected in common human malignancies not associated with NF2, including colorectal and lung cancer. Furthermore, tumors without NF2 mutations and with unaltered NF2 transcript levels, but with low Merlin expression, have been reported. The present study demonstrated that NF2 is also regulated by microRNAs (miRNAs) through direct interaction with evolutionarily conserved miRNA response elements (MREs) within its 3'‑untranslated region (3'UTR). Dual‑Luciferase assays in human colorectal carcinoma (HCT116) and lung adenocarcinoma (A549) cells revealed downregulation of NF2 by miR‑92a‑3p via its wild‑type 3'UTR, but not NF2‑3'UTR with mutated miR‑92a‑3p MRE. HCT116 cells overexpressing miR‑92a‑3p exhibited significant downregulation of endogenous NF2 mRNA and protein levels, which was rescued by co‑transfection of a target protector oligonucleotide specific for the miR‑92a‑3p binding site within NF2‑3'UTR. miR‑92a‑3p overexpression in HCT116 and A549 cells promoted migration, proliferation and resistance to apoptosis, as well as altered F‑actin organization compared with controls. Knockdown of NF2 by siRNA phenocopied the oncogenic effects of miR‑92a overexpression on HCT116 and A549 cells. Collectively, the findings of the present study provide functional proof of the unappreciated role of miRNAs in NF2 regulation and tumor progression, leading to enhanced oncogenicity.
Full text link https://tinyurl.com/2ax5x7yn