Biology subjects

Rangarajan, A.

Publications and source records attributed to Rangarajan, A..

10 recordsLinked to original sources

bioRxiv2025

Stromal LOX/FAK/beta catenin pathway locks mammary fibroblasts into a tumor-promoting myCAF state

BackgroundCancer-associated fibroblasts (CAFs) sustain tumor progression, yet the soluble cues that maintain their myofibroblast (myCAF) state are poorly defined. Transforming growth factor beta (TGF-{beta}) is a canonical CAF activator. This study aims to identify TGF-{beta}-induced secreted mediators that reinforce the myCAF phenotype in breast cancer and map the downstream signaling cascade. Methods and ResultsSecretome profiling of primary patient-derived myCAFs and human mammary fibroblasts (HMF3s) engineered to over-express TGF-{beta}1 revealed 20 extracellular-matrix remodelers shared exclusively by both activated states; lysyl oxidase (LOX) was the top-ranked hit. LOX knockdown abrogated TGF-{beta}-driven -smooth-muscle actin (-SMA) induction, collagen-gel contraction and migration in HMF3s, and reduced constitutive -SMA and {beta}-catenin in myCAFs. Mechanistically, TGF-{beta} upregulated LOX, which activated focal-adhesion kinase (FAK), leading to p38 MAPK- and Akt-mediated Ser9 phosphorylation (inactivation) of GSK3{beta} and consequent {beta}-catenin stabilization. In HCC1806-luciferase orthotopic xenografts, CAFs accelerated tumor growth, whereas LOX-deficient CAFs lost this pro-tumoral effect. ConclusionLOX is a pivotal autocrine effector of TGF-{beta} that locks breast CAFs into a pro-tumoral myCAF state through a LOX/FAK/GSK3{beta}/{beta}-catenin axis. Targeting stromal LOX may disrupt CAF activation and curb breast cancer progression.

cancer biology↗

bioRxiv2025

Balance between autophagy and apoptosis determines anoikis resistance: a mathematical model on cell fate

Although several cancer cells are shed into circulation, most die due to stress, including loss of matrix attachment which triggers anoikis. A subset, however, acquires anoikis resistance, and survives to seed metastasis. How matrix-deprived cancer cells balance autophagy and apoptosis to decide cell fate remains unclear. Here, we formulated a deterministic ordinary differential equation (ODE)-based model of Akt-AMPK-ERK signal interaction to study this balance. Simulations revealed that high AMPK activity, with heterogenous ERK and low pAkt levels promote survival, whereas high pAkt shifts towards apoptosis. Molecular perturbation analyses revealed a signaling hierarchy in which Akt dominates over AMPK which further dominates over ERK. This model further predicts that the extent of autophagy determines apoptotic outcomes with high autophagy favoring survival of matrix-detached MDA-MB-231 cells. Together, our study provides a mathematical framework linking signaling dynamics to cell-fate decisions governing anoikis resistance. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=147 HEIGHT=200 SRC="FIGDIR/small/658583v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@d40fdorg.highwire.dtl.DTLVardef@25a839org.highwire.dtl.DTLVardef@475401org.highwire.dtl.DTLVardef@dc4e6b_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2025

A structure-based tool to interpret the significance of kinase mutations in clinical next generation sequencing in cancer

IntroductionClinical workflows to analyze variants of unknown significance (VUSs) found in clinical next generation sequencing (NGS) are labor intensive, requiring manual analysis of published data for each variant. There is a strong need for tools and resources that provide a consistent way to analyze variants. With the explosion of clinical NGS data and the concurrent availability of protein structures through the Protein Data Bank and protein models through programs such as AlphaFold, there exists an unprecedented opportunity to use structural information to help standardize NGS analysis with the overall goal of advancing personalized cancer therapy. MethodsUsing the Catalogue of Somatic Mutations in Cancer (COSMIC), the largest curated database of clinical cancer mutations, we mapped thousands of missense mutations in the kinase and juxtamembrane (JM) domains of 48 receptor tyrosine kinases (RTKs) onto structurally aligned kinase structures, then clustered known activating mutations along with VUSs based on proximity in three-dimensional structure. Using cell-based models we illustrate that our resource can be used to identify activating mutations and provide insight into mechanisms of kinase activation and regulation. ResultsWe provide a database of structurally aligned and functionally annotated mutations that can be used as a tool to evaluate kinase VUSs based on their structural alignment with known activating mutations. The tool can be accessed through a user-friendly website in which one can input a kinase mutation of interest, and the system will output a list of structurally analogous mutations in other kinases, as well as their functional annotations. DiscussionWe expect our database to be an important addition to the current tools and resources used to analyze clinical NGS, with important clinical implications to guide recommendations for personalized cancer therapy.

cancer biology↗

bioRxiv2024

Combined analysis of somatic mutations and gene expression reveals nuclear speckles-associated enhanced stemness in gingivobuccal carcinoma under DNA damage response

Smokeless tobacco chewing habits in India lead to a high prevalence of Gingivobuccal oral squamous cell carcinoma (OSCC-GB). Cancer stem cells (CSCs) are a sub-population of cancer cells within a tumor with stem-like properties and are believed to contribute to tumor initiation, progression, increased resistance to drug therapy, and promote post-therapeutic cancer relapse. An RNA-seq data-based integrative analysis of somatic mutation and gene expression was performed to explore the role of CSCs in disease progression using the novel Indian-origin OSCC-GB cell line IIOC019 from a patient with tobacco-chewing habit. The identified DNA damage-related known mutational signature (ID1, SBS5) indicates the impact of smokeless tobacco-related carcinogens in the IIOC019 cell line. The differentially expressed somatic variants, functional impact predictions, and survival analysis reveal the role of DNA damage response (DDR)-related genes in OSCC-GB, with the SON gene as a significant player. The study suggests that the loss of function in a somatic variant of the SON gene is associated with enhanced stemness and increased risk of disease progression in OSCC-GB under DDR conditions. The newly identified CSC-associated somatic variants appear to promote cancer spread, local recurrence, and resistance to chemotherapy or radiotherapy, contributing to the high mortality rates among Indian OSCC-GB patients.

genomics↗

bioRxiv2022

Prolactin-induced AMPK stabilizes alveologenesis and lactogenesis through regulation of STAT5 signaling

AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that regulates energy homeostasis at cellular and organismal levels. It has been shown to affect several steps of breast cancer progression in a context-dependent manner. However, its role in normal mammary gland development and physiology remains ill-explored. Here, we show that AMPK expression and activity increased within murine mammary epithelia from puberty to pregnancy with highest levels during lactation, and then declined during involution. In ex vivo cultures of mammary epithelial cells (MECs) in organotypic scaffolds, treatment with lactogenic hormone prolactin (PRL) enhanced AMPK expression and activity. To understand the role of AMPK on mammary morphogenesis in vivo, we generated mice with conditional knockout of AMPK isoforms 1 and 2 (AMPK KO) in MECs. AMPK KO mammary glands showed accelerated alveolar development with increased epithelial content of both luminal and myoepithelial lineages, suggestive of hyperproliferation. AMPK KO mice also showed elevated beta-casein expression during pregnancy and lactation. These observations were phenocopied upon treatment of ex vivo cultivated wild-type MECs with a cognate AMPK inhibitor. AMPK null MECs showed increased phosphorylated STAT5 which is known to drive alveologenesis downstream of prolactin signaling. Our study identifies a novel interplay between AMPK and PRL-STAT5 signaling that determines mammary alveologenesis and differentiation.

developmental biology↗

bioRxiv2021

An acetyl-click screening platform identifies a small molecule inhibitor of Histone Acetyltransferase 1 (HAT1) with anti-tumor activity

HAT1 is a central regulator of chromatin synthesis that acetylates nascent histone H3:H4 tetramers in the cytoplasm. It may have a role in cancer metabolism by linking cytoplasmic production of acetyl-CoA to nuclear acetyl flux. This is because the HAT1 di-acetylation mark is not propagated in chromatin and instead is de-acetylated after nascent histone insertion into chromatin. Thus, HAT1 likely provides a nuclear source of free acetate that may be recycled to acetyl-CoA for nuclear acetylation reactions. Correspondingly, suppression of HAT1 protein expression impairs tumor growth. To ascertain whether targeting HAT1 is a viable anti-cancer treatment strategy we sought to identify small molecule inhibitors of HAT1. We developed a high-throughput HAT1 acetyl-click assay to facilitate drug discovery and enzymology. Screening of small molecules computationally predicted to bind the active site led to the discovery of multiple riboflavin analogs that inhibited HAT1 enzymatic activity by competing with acetyl-CoA binding. These hits were refined by synthesis and testing over 70 analogs, which yielded structure-activity relationships. The isoalloxazine core was required for enzymatic inhibition, whereas modifications of the ribityl sidechain improved enzymatic potency and cellular growth suppression. These efforts resulted in a lead compound (JG-2016) that suppressed growth of human cancer cells lines in vitro and impaired tumor growth in vivo. This is the first report of a small molecule inhibitor of the HAT1 enzyme complex and represents a step towards targeting this pathway for cancer therapy.

biochemistry↗

bioRxiv2020

Multistability and consequent phenotypic plasticity in AMPK-Akt double negative feedback loop in cancer cells

Adaptation and survival of cancer cells to various stress and growth factor conditions is crucial for successful metastasis. A double-negative feedback loop between two serine/threonine kinases AMPK and Akt can regulate the adaptation of breast cancer cells to matrix-deprivation stress. This feedback loop can generate majorly two phenotypes or cell states: matrix detachment-triggered pAMPKhigh/ pAktlow state, and matrix (re)attachment-triggered pAkthigh/ pAMPKlow state. However, whether these two cell states can exhibit phenotypic plasticity and heterogeneity in a given cell population, i.e., whether they can co-exist and undergo spontaneous switching to generate the other subpopulation, remains unclear. Here, we develop a mechanism-based mathematical model that captures the set of experimentally reported interactions among AMPK and Akt. Our simulations suggest that the AMPK-Akt feedback loop can give rise to two co-existing phenotypes (pAkthigh/ pAMPKlow and pAMPKhigh/pAktlow) in specific parameter regimes. Next, to test the model predictions, we segregated these two subpopulations in MDA-MB-231 cells and observed that each of them was capable of switching to another in adherent conditions. Finally, the predicted trends are supported by clinical data analysis of TCGA breast cancer and pan-cancer cohorts that revealed negatively correlated pAMPK and pAkt protein levels. Overall, our integrated computational-experimental approach unravels that AMPK-Akt feedback loop can generate multistability and drive phenotypic switching and heterogeneity in a cancer cell population.

cancer biology↗

bioRxiv2020

Epigenetic feedback and stochastic partitioning during cell division can drive resistance to EMT

Epithelial-mesenchymal transition (EMT) and its reverse process mesenchymal-epithelial transition (MET) are central to metastatic aggressiveness and therapy resistance in solid tumors. While molecular determinants of both processes have been extensively characterized, the heterogeneity in the response of tumor cells to EMT and MET inducers has come into focus recently, and has been implicated in the failure of anti-cancer therapies. Recent experimental studies have shown that some cells can undergo an irreversible EMT depending on the duration of exposure to EMT-inducing signals. While the irreversibility of MET, or equivalently, resistance to EMT, has not been studied in as much detail, evidence supporting such behavior is slowly emerging. Here, we identify two possible mechanisms that can underlie resistance of cells to undergo EMT: epigenetic feedback in ZEB1/GRHL2 feedback loop and stochastic partitioning of biomolecules during cell division. Identifying the ZEB1/GRHL2 axis as a key determinant of epithelial-mesenchymal plasticity across many cancer types, we use mechanistic mathematical models to show how GRHL2 can be involved in both the abovementioned processes, thus driving an irreversible MET. Our study highlights how an isogenic population may contain subpopulation with varying degrees of susceptibility or resistance to EMT, and proposes a next set of questions for detailed experimental studies characterizing the irreversibility of MET/resistance to EMT.

cancer biology↗

bioRxiv2019

Feedback loops involving AMPK, ERK and TFEB in matrix detachment leads to non-genetic heterogeneity

Cancer cells metastasize by evading apoptosis induced due to the detachment from the extracellular matrix. Deciphering the adaptive strategies employed by cancer cells in matrix-deprived condition can help design novel therapeutic strategies to tackle metastasis. Here, we provide evidence for non-genetic heterogeneity in matrix-detached cells enabled by feedback loop among AMPK, ERK and TFEB that determines autophagy maturation and cell survival. The subpopulation of matrix-detached cells with pAMPKlow/pERKhigh/TFEBlow state show autophagy maturation arrest and elevated cell death. Conversely, pAMPKhigh/pERKlow/ TFEBhigh cells show high autophagy maturation and better cell survival. We show that AMPK inhibits ERK activity in suspension; ERK negatively regulates TFEB which promotes autophagy maturation and re-enforces AMPK. Inhibition of ERK promotes autophagy maturation, cell survival and metastasis in vivo, while AMPK inhibition (and TFEB depletion) renders the population homogeneous by depleting the pAMPKhigh/pERKlow/TFEBhigh subpopulation, driving detachment-induced death. Such non-genetic heterogeneity is further deciphered by mathematical modelling and RNA-sequencing data of circulating tumor cells (CTCs) isolated from breast cancer patients. Altogether, our work unravels a contextual feedback loop involving two kinases and a transcription factor that helps a subpopulation to evade cell death in matrix-deprived condition. Disrupting such feedback loops may offer improved therapeutic efficacy and a novel approach to constrain metastasis. Significance of the StudyAttachment to the extracellular matrix is pivotal for the growth and survival of normal epithelial cells. In contrast, cancer cells acquest the ability to survive matrix-deprivation and cause cancer spread, or metastasis, which is a leading cause of cancer-related deaths. Non-genetic heterogeneity within cancer cell populations is increasingly being recognized as a major cause of treatment failure. However, the implications of such heterogeneity in the survival of matrix-detached cancer cells remain poorly understood. In this study, we demonstrate feedback loops involving kinases and transcription factor to maintain non-genetic heterogeneous population, where population harbouring pAMPKhigh/pERKlow/TFEBhigh status shows survival advantage. Targeting such feedback loops that generate non-genetic heterogeneity can open newer therapeutic means to restrict cancer spread.

cancer biology↗

bioRxiv2018

AMPK promotes Notch1 stability to potentiate hypoxia-induced breast cancer stemness and drug resistance

Hypoxia is a hall mark of solid tumor microenvironment and contributes to tumor progression and therapy failure. The developmentally important Notch pathway is implicated in cellular response of cancer cells to hypoxia. Yet, the mechanisms that potentiate Notch signaling under hypoxia are not fully understood. Hypoxia is also a stimulus for AMP-activated protein kinase (AMPK), a major cellular energy sensor. In this study, we investigated if AMPK interacts with the Notch pathway and influences the hypoxia-response of breast cancer cells. Activating AMPK with pharmacological agent or genetic approaches led to an increase in the levels of cleaved Notch1 and elevated Notch signaling in invasive breast cancer cell lines. In contrast, inhibition or depletion of AMPK reduced the amount of cleaved Notch1. Significantly, we show that the hypoxia-induced increase in cleaved Notch1 levels requires AMPK activation. Probing into the mechanism, we demonstrate that AMPK activation impairs the interaction between cleaved Notch1 and its ubiquitin ligase, Itch/AIP4 through the tyrosine kinase Fyn. Under hypoxia, the AMPK-Fyn axis promotes inhibitory phosphorylation of Itch which abrogates its interaction with substrates, thus stabilizing cleaved Notch1 by reducing its ubiquitination and degradation. We further show that inhibition of AMPK alleviates the hypoxia-triggered, Notch-mediated stemness and drug resistance phenotype. Breast cancer patient samples also showed co-expression of hypoxia/AMPK/Notch gene signature. Our work thus establishes AMPK as a key component in the adaptation of breast cancer cells to hypoxia, and proposes therapeutic inhibition of AMPK to mitigate the hypoxia-triggered aggressiveness.

cancer biology↗