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Ranjan, G.

Publications and source records attributed to Ranjan, G..

10 recordsLinked to original sources
bioRxiv2025

Orai3 orchestrates gemcitabine resistance in pancreatic cancer via NFATc1-SLIT3 axis

Pancreatic Cancer (PC) is one of the most aggressive cancers and is associated with poor prognosis. Gemcitabine is the first-line chemotherapy for PC. While gemcitabine-based regimes offer survival benefits, the acquired gemcitabine resistance leads to reoccurrence, metastasis and the long-term survival rate remains dismal. Although there is substantial clinical evidence for gemcitabine resistance, the cellular and molecular mechanisms driving gemcitabine resistance remain largely unappreciated. Here, we reveal that Orai3, a Ca2+ selective channel, is a crucial driver of gemcitabine resistance. We demonstrate that Orai3 is upregulated in gemcitabine-resistant PC cells. Orai3 silencing in these cells decreases proliferation, induces cell cycle arrest, enhances apoptosis, and moderates stemness characteristics. Notably, studies in zebrafish model corroborate the significance of Orai3 in gemcitabine resistance in vivo. Mechanistically, our unbiased RNA-seq analysis coupled with robust functional studies show that SLIT3 works downstream of Orai3 to drive gemcitabine resistance. Finally, we report that NFATc1 transcription factor bridges Orai3 to SLIT3 transcription. Taken together, this study identifies Orai3 as a key orchestrator of gemcitabine resistance and uncovers a unique Orai3-NFATc1-SLIT3 signaling module that drives chemoresistance. Hence, this work reveals Orai3 as a promising target for synergistic therapeutic approach to combat chemoresistance. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/671673v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@16fea2org.highwire.dtl.DTLVardef@1a77a3forg.highwire.dtl.DTLVardef@4aef28org.highwire.dtl.DTLVardef@19b62a3_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights{Rightarrow} Orai3 is upregulated in gemcitabine-resistant pancreatic cancer cells {Rightarrow}Orai3 is essential for chemo-sensitivity, migration, invasion and stemness in vitro {Rightarrow}Orai3 regulates metastasis of gemcitabine-resistant pancreatic cancer cells in vivo {Rightarrow}Unbiased RNA-seq reveals that SLIT3 functions downstream of Orai3 to drive gemcitabine resistance {Rightarrow}NFATc1 transcription factor bridges Orai3 to SLIT3 transcription

cancer biology↗
bioRxiv2024

NFAT2 drives both Orai3 transcription and protein degradation by harnessing the differences in epigenetic landscape

The functional significance of any protein in physiological processes and pathological conditions is largely dependent on its expression profile. Therefore, nature has evolved several autonomous mechanisms to regulate protein expression such as transcription, translation, post-translational modifications and epigenetic changes. These processes are typically controlled by distinct molecular players with no overlapping roles. Here, we reveal that same transcription factor, NFAT2 regulates both transcription and lysosomal degradation of Orai3 oncochannel in a context dependent manner. We demonstrate that NFAT2 drives Orai3 transcription and thereby increases Orai3 levels in non-metastatic cancerous cells. While in invasive and metastatic cancerous cells, NFAT2 induces Orai3 lysosomal degradation by transcriptionally enhancing the levels of MARCH8 E3 ubiquitin ligase. Our biochemical and super-resolution microscopy data show that MARCH8 physically interacts with Orai3 eventually resulting in its degradation. Mechanistically, the dichotomy in regulation of Orai3 expression emerges from the differences in the epigenetic landscape of MARCH8. We uncover that the MARCH8 promoter is highly methylated in non-metastatic cancerous cells and hence NFAT2 does not induce MARCH8 mediated Orai3 degradation in these cells. Importantly, we demonstrate that MARCH8 restricts pancreatic cancer metastasis by targeting Orai3 degradation thereby highlighting pathophysiological importance of this signaling module. Taken together, we report a unique and clinically relevant scenario wherein nature has commissioned the same transcription factor to both enhance and curtail the expression of a target protein. Highlights[tpltrtarr] NFAT2 transcriptionally upregulates Orai3 Ca2+ channel in non-metastatic cells [tpltrtarr]NFAT2 induces Orai3 lysosomal degradation via MARCH8 E3 ubiquitin ligase in metastatic cells [tpltrtarr]The dichotomy in NFAT2s function is an outcome of differences in the methylation status of MARCH8 promoter [tpltrtarr]MARCH8 inhibits pancreatic cancer metastasis by driving Orai3 degradation Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/628081v2_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1292480org.highwire.dtl.DTLVardef@1b5b61org.highwire.dtl.DTLVardef@c4483org.highwire.dtl.DTLVardef@16607f8_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗
bioRxiv2024

LncRNA TAAL is a Modulator of Tie1-Mediated Vascular Function in Diabetic Retinopathy

Diabetic retinopathy (DR), a leading cause of vision impairment and blindness, is characterized by abnormal retinal vascular changes due to chronic hyperglycemia. The Tie-1 signaling pathway, essential for vascular growth and remodeling, has emerged as a key therapeutic target, though its molecular mechanisms and interactome remain largely unclear. Through a protein-centric approach, we identified a novel lncRNA and named it Tie1-associated angiogenic lncRNA (TAAL). TAAL lncRNA regulates endothelial cell migration, proliferation, tube formation, and permeability by modulating ER-calcium homeostasis and cytoskeleton dynamics. In zebrafish, taal modulation led to angiogenic defects, which were rescued by human TAAL orthologue. Our molecular studies further revealed that TAAL negatively regulates Tie1 protein via ubiquitin-mediated degradation. Notably, TAAL expression is upregulated in the blood of DR patients and downregulated in endothelial DR cell models. Overexpression of TAAL restored endothelial permeability and VE-cadherin surface expression. These findings establish TAAL as a novel regulator of Tie1 protein turnover, with potential therapeutic implications for diabetic retinopathy.

genetics↗
bioRxiv2024

SCAR-6 elncRNA locus epigenetically regulates PROZ and modulates coagulation and vascular function

Syntenic conservation is an effective strategy to identify evolutionarily conserved lncRNA orthologs. In this study, we identified a novel uncharacterized conserved lncRNA known as Syntenic Cardiovascular Conserved Region-Associated lncRNA-6 (scar-6) and functionally validated its role in coagulation and cardiovascular function. Precise editing of the scar-6 lncRNA locus in zebrafish (scar-6gib007{Delta}12/{Delta}12) resulted in cranial hemorrhage and permeability defects. Further analysis, including overexpression, locus editing, and rescue experiments, provided compelling evidence for the critical role of the scar-6 transcript in the coagulation process of zebrafish. Notably, rescue attempts were unsuccessful in mitigating cranial hemorrhage. Molecular investigation revealed that the scar-6 RNA acts as an enhancer lncRNA (elncRNA), and controls the expression of prozb, an inhibitor of factor Xa, through the enhancer element on its locus. The scar-6 locus actively suppresses the loop formation between prozb and scar-6 sequences, facilitated by methylation of CpG island via the prdm14-PRC2 complex, which is stabilized by the scar-6 elncRNA transcript. Disruption of this mechanism in scar-6gib007{Delta}12/{Delta}12 zebrafish led to impaired vascular function and subsequent hemorrhage. This was triggered by the activation of the PAR2 receptor mediated by upregulation of prozb, which in turn caused NF-{kappa}B-mediated endothelial cell activation. This study presents novel evidence for the multifaceted function of the scar-6 locus, highlighting its crucial role in regulating the coagulation cascade gene prozb and maintaining homeostasis and vascular function. Synopsis O_FIG O_LINKSMALLFIG WIDTH=177 HEIGHT=200 SRC="FIGDIR/small/604148v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@ea55e4org.highwire.dtl.DTLVardef@ad01beorg.highwire.dtl.DTLVardef@4da1eborg.highwire.dtl.DTLVardef@c153a9_HPS_FORMAT_FIGEXP M_FIG C_FIG ProZ-PZI is a natural inhibitor of activated coagulation factor X (F10) and plays a major role in maintaining hemostasis in-vivo. Here, the novel evolutionary syntenic conserved scar-6 elncRNA locus is shown to regulate prozb expression and control coagulation and vascular integrity in zebrafish. O_LIThe scar-6 acts as an enhancer lncRNA (elncRNA). It controls prozb expression and modulates coagulation and vascular function in zebrafish. C_LIO_LIThe scar-6 elncRNA stabilizes the Prdm14-PRC2 complex binding to scar-6 locus. This inhibits prozb/scar-6 looping via methylating the CpG island under wildtype conditions. C_LIO_LIOverexpressed prozb in scar-6 edited animals activates PAR2 receptor, causing endothelial cell activation and vascular dysfunction. C_LI

genetics↗
bioRxiv2024

Syntenic lncRNAs exhibit DNA regulatory functions with sequence evolution

Syntenic long non-coding RNAs (lncRNAs) often show limited sequence conservation across species, prompting concern in the field. This study delves into functional signatures of syntenic lncRNAs between humans and zebrafish. Syntenic lncRNAs have high expression in zebrafish and [~]90% near protein-coding genes in sense or antisense orientation. During early zebrafish development and human embryonic stem cells (H1-hESC), are enriched with cis-regulatory repressor signatures, influencing development-associated genes. In later zebrafish developmental stages and specific human cell lines, these lncRNAs serve as enhancers or transcription-start-sites(TSS) for protein-coding. Analysis of Transposable Elements (TEs) in syntenic lncRNA sequence divergence unveils intriguing patterns, human lncRNAs show enrichment in simple repeat elements, while zebrafish counterparts exhibit LTR element enrichment. This sequence evolution, possibly stemming from post-rearrangement mutations, enhances DNA elements or cis-regulatory functions. It may also contribute to vertebrate innovation by creating novel TF binding sites within the locus. This study sheds light on the conserved functionality of syntenic lncRNAs through DNA elements, emphasizing their role across species despite sequence divergence.

genetics↗
bioRxiv2023

Mitochondrial calcium signaling mediated transcriptional regulation of keratin filaments is a critical determinant of melanogenesis

Mitochondria are versatile organelles that regulate several physiological functions. Many mitochondria-controlled processes are driven by mitochondrial Ca2+ signaling. However, role of mitochondrial Ca2+ signaling in melanosome biology remains unknown. Here, we show that pigmentation requires mitochondrial Ca2+ uptake. In vitro gain and loss of function studies demonstrated that Mitochondrial Ca2+ Uniporter (MCU) is crucial for melanogenesis while the MCU rheostats, MCUb and MICU1 negatively control melanogenesis. Zebrafish and mouse models showed that MCU plays a vital role in pigmentation in vivo. Mechanistically, MCU controls activation of transcription factor NFAT2 to induce expression of three keratins (keratin 5, 7 and 8), which we report as positive regulators of melanogenesis. Interestingly, keratin 5 in turn modulates mitochondrial Ca2+ uptake thereby this signaling module acts as a negative feedback loop that fine-tunes both mitochondrial Ca2+ signaling and melanogenesis. Mitoxantrone, an FDA approved drug that inhibits MCU, decreases physiological melanogenesis. Collectively, our data demonstrates a critical role for mitochondrial Ca2+ signaling in vertebrate pigmentation and reveal the therapeutic potential of targeting MCU for clinical management of pigmentary disorders. Given the centrality of mitochondrial Ca2+ signaling and keratin filaments in cellular physiology, this feedback loop may be functional in a variety of other pathophysiological conditions. HighlightsO_LIMCU complex mediated mitochondrial Ca2+ uptake is a novel regulator of vertebrate pigmentation C_LIO_LIKeratin filaments bridge mitochondrial Ca2+ signaling to melanosome biogenesis and maturation C_LIO_LITranscription factor NFAT2 connects mitochondrial Ca2+ dynamics to keratins expression C_LIO_LIMCU-NFAT2-Keratin 5 signaling module generates a negative feedback loop to maintain mitochondrial Ca2+ homeostasis and to ensure optimal melanogenesis C_LIO_LIInhibiting MCU with mitoxantrone, an FDA approved drug, leads to reduction in physiological pigmentation C_LI

cell biology↗
bioRxiv2021

Insights from Genomes and Genetic Epidemiology of SARS-CoV-2 isolates from the state of Andhra Pradesh

Coronavirus disease (COVID-19) emerged from a city in China and has now spread as a global pandemic affecting millions of individuals. The causative agent, SARS-CoV-2 is being extensively studied in terms of its genetic epidemiology using genomic approaches. Andhra Pradesh is one of the major states of India with the third-largest number of COVID-19 cases with limited understanding of its genetic epidemiology. In this study, we have sequenced 293 SARS-CoV-2 genome isolates from Andhra Pradesh with a mean coverage of 13,324X. We identified 564 high-quality SARS-CoV-2 variants, out of which 15 are novel. A total of 18 variants mapped to RT-PCR primer/probe sites, and 4 variants are known to be associated with an increase in infectivity. Phylogenetic analysis of the genomes revealed the circulating SARS-CoV-2 in Andhra Pradesh majorly clustered under the clade A2a (94%), while 6% fall under the I/A3i clade, a clade previously defined to be present in large numbers in India. To the best of our knowledge, this is the most comprehensive genetic epidemiological analysis performed for the state of Andhra Pradesh.

genomics↗
bioRxiv2020

Initial insights into the genetic epidemiology of SARS-CoV-2 isolates from Kerala suggest local spread from limited introductions

Coronavirus disease 2019 (COVID-19) rapidly spread from a city in China to almost every country in the world, affecting millions of individuals. Genomic approaches have been extensively used to understand the evolution and epidemiology of SARS-CoV-2 across the world. Kerala is a unique state in India well connected with the rest of the world through a large number of expatriates, trade, and tourism. The first case of COVID-19 in India was reported in Kerala in January 2020, during the initial days of the pandemic. The rapid increase in the COVID-19 cases in the state of Kerala has necessitated the understanding of the genetic epidemiology of circulating virus, evolution, and mutations in SARS-CoV-2. We sequenced a total of 200 samples from patients at a tertiary hospital in Kerala using COVIDSeq protocol at a mean coverage of 7,755X. The analysis identified 166 unique high-quality variants encompassing 4 novel variants and 89 new variants identified for the first time in SARS-CoV-2 samples isolated from India. Phylogenetic and haplotype analysis revealed that the circulating population of the virus was dominated (94.6% of genomes) by three distinct introductions followed by local spread, apart from identifying polytomies suggesting recent outbreaks. The genomes formed a monophyletic distribution exclusively mapping to the A2a clade. Further analysis of the functional variants revealed two variants in the S gene of the virus reportedly associated with increased infectivity and 5 variants that mapped to five primer/probe binding sites that could potentially compromise the efficacy of RT-PCR detection. To the best of our knowledge, this is the first and most comprehensive report of genetic epidemiology and evolution of SARS-CoV-2 isolates from Kerala.

genomics↗
bioRxiv2020

High throughput detection and genetic epidemiology of SARS-CoV-2 using COVIDSeq next generation sequencing

The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance and for determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for detection of SARS-CoV-2, with an additional advantage of enabling genetic epidemiology of SARS-CoV-2.

genomics↗