Biology subjects

Hempel, N.

Publications and source records attributed to Hempel, N..

At least 19 recordsLinked to original sources

bioRxiv2026

A Quartet of Native Orai Channel Isoforms Orchestrates Graded NFAT Activation and Transcription

The Ca{superscript 2} release-activated Ca{superscript 2} (CRAC) channel mediates store-operated calcium entry (SOCE), a ubiquitous pathway essential for many cell types, including immune cells. Three Orai (Orai1/2/3) proteins constitute the plasma membrane pore-forming units of CRAC channels that are activated by the endoplasmic reticulum (ER) Ca2+-sensing STIM1/2 proteins when ER Ca2+ stores are depleted. Orai1/2/3 are differentially expressed across primary cells with discernible differences in their structures and biophysical properties. Further, Orai1 has two alternatively translated isoforms: long mammalian-specific Orai1 and the 63-residue shorter Orai1{beta}, which is evolutionarily older and conserved across vertebrates. Whether Orai1/1{beta}/2/3 produce unique cytosolic Ca{superscript 2} signatures that bias transcriptional responses through effectors like NFAT is unclear. Here, we used HEK293 cells engineered to express one native Orai isoform and show that all Orai isoforms couple to NFAT1/4 induction. The magnitude of NFAT1/4 induction for each Orai isoform matches that of SOCE, with the following profile: Orai1{beta}>Orai1>>Orai2>Orai3. Near-native re-expression of either Orai1 or Orai1{beta} in primary murine Orai1-/- CD4 T cells restored SOCE, NFAT activation, cytokine production and promoted near identical transcriptional responses enriched for immune activation pathways. An analysis of genetic and clinical data of human individuals showed that homozygous null mutations selectively abolishing Orai1 are not associated with disease resembling CRAC channelopathy. Primary T cells from individuals homozygous or heterozygous for an Orai1 null mutation showed enhanced, rather than impaired, SOCE and NFAT induction. Our data indicate that NFAT activation and transcriptional outputs are primarily driven by the graded strength of SOCE mediated by each isoform of the Orai quartet.

cell biology↗

bioRxiv2026

Loss of the lncRNA SOX1-OT promotes p53-dependent cell-cycle arrest in astrocytes

Long non-coding RNAs (lncRNAs) are increasingly recognized as regulators of brain cell function, but their roles in astrocyte biology and neurodegeneration remain poorly understood. Here, we identify Sox1ot/SOX1-OT as a conserved, brain-enriched lncRNA that is downregulated in Alzheimers disease and in reactive astrocyte states. Antisense oligonucleotide-mediated depletion of Sox1ot in astrocytes revealed a transcriptional program marked by activation of p53 target genes selectively associated with cell-cycle inhibitory pathways. Consistent with this, Sox1ot depletion enhanced p53 occupancy at target promoters such as Cdkn1a, increased Cdkn1a expression and levels of its protein product p21, and thereby induced G1 arrest and reduced astrocyte proliferation. In contrast, other canonical p53 outputs, including apoptosis and senescence, were not affected, indicating that Sox1ot selectively modulates distinct branches of p53 signaling. Notably, loss of Sox1ot/SOX1-OT was accompanied by impaired glutamate uptake, reduced lactate secretion, and altered astrocyte support functions, suggesting that these deficits arise as downstream consequences of the p53-dependent transcriptional shift rather than direct primary effects of Sox1ot loss. Together, these findings identify SOX1-OT as an astrocyte-enriched regulatory layer that constrains a p53-dependent cell-cycle program and highlight its role in shaping astrocyte state transitions in Alzheimers disease.

neuroscience↗

bioRxiv2025

RHOV is a Detachment-Responsive Rho GTPase Necessary for Ovarian Cancer Peritoneal Metastasis

All ovarian cancer subtypes spread via transcoelomic metastasis, where cells disseminate into the peritoneal fluid, resist anoikis, and form multicellular aggregates that invade the peritoneum. This represents the main driver of morbidity and mortality for peritoneal cancer patients. Mechanisms necessary for cancer cells to survive matrix detachment and initiate transcoelomic metastasis remain poorly defined. To address this, we identified a conserved detachment-sensitive gene signature activated shortly after matrix-detachment across multiple ascites-derived cancer cell lines. RHOV, an atypical, constitutively active and understudied member of the Rho GTPase family, emerged as a top upregulated transcript, which was confirmed in patient ascites-derived tumor cells. Functionally, loss of RHOV impairs anoikis resistance, multicellular aggregate integrity, migration and invasion, and completely abolishes transcoelomic tumor progression in vivo. RHOV enhances c-Jun signaling and cytoskeletal remodeling, which is dependent on both RHOV GTP-binding and membrane localization. These findings define RHOV as a novel detachment-sensitive Rho GTPase and establish RHOV as a critical regulator of peritoneal metastasis for the first time.

cancer biology↗

bioRxiv2025

CDKN2ALow cancer cells outcompete macrophages for microenvironmental zinc to drive immunotherapy resistance

Anti-PD1 therapies are primarily thought to rely on functional T cell responses; yet tumors with limited T cell infiltration can still benefit, suggesting alternative mechanisms contribute to therapeutic efficacy. Indeed, we found that myeloid-rich, T cell-poor tumor models respond to anti-Pd1, and this is dependent on a cancer cell-macrophage crosstalk mediated by cancer cell Cdkn2a expression. Mechanistically, we found that cancer cells with decreased Cdkn2a expression (Cdkn2aLow), which occurs in [~]50% of all human cancers, reorganize zinc compartmentalization by upregulating the zinc importer Slc39a9 at the plasma membrane. Increased cancer cell plasma membrane Slc39a9 leads to intracellular zinc accumulation in cancer cells and depletion of zinc in the tumor microenvironment (TME), resulting in zinc-starved tumor-associated macrophages (TAMs) with reduced phagocytic activity. Restoring zinc availability in TAMs--via dietary supplementation or Slc39a9 knockdown in cancer cells--reprograms TAMs to a pro-phagocytic state and sensitizes Cdkn2aLow tumors to anti-Pd1 therapy. Remarkably, Slc39a9 knockdown tumors respond to anti-Pd1 in Rag1-/- mice, and co-injection of zinc-replete macrophages is sufficient to drive an anti-Pd1 response in immunodeficient mice, demonstrating the T cell-independent nature of this response. Clinically, TAMs from CDKN2ALow cancer patients show reduced zinc and phagocytosis gene signatures. Moreover, patients with lower circulating zinc levels have significantly worse time-to-event outcomes than those with higher levels. Together, these findings uncover a previously unrecognized mechanism by which Cdkn2aLow cancer cells outcompete TAMs for zinc, impairing their function and limiting anti-Pd1 efficacy. They also provide evidence that macrophages alone, without T cells, can enhance anti-PD1 response through zinc-mediated reprogramming of phagocytosis.

cancer biology↗

bioRxiv2024

Loss of the predicted cell adhesion molecule MPZL3 promotes EMT and chemoresistance in ovarian cancer

Myelin protein zero-like 3 (MPZL3) is an Immunoglobulin-containing transmembrane protein with predicted cell adhesion molecule function. Loss of 11q23, where the MPZL3 gene resides, is frequently observed in cancer, and MPZL3 copy number alterations are frequently detected in tumor specimens. Yet the role and consequences of altered MPZL3 expression have not been explored in tumor development and progression. We addressed this in ovarian cancer, where both MPZL3 amplification and deletions are observed in respective subsets of high-grade serous specimens. While high and low MPZL3 expressing populations were similarly observed in primary ovarian tumors from an independent patient cohort, metastatic omental tumors largely displayed decreased MPZL3 expression, suggesting that MPZL3 loss is associated with metastatic progression. MPZL3 knock-down leads to strong upregulation of vimentin and an EMT gene signature that is associated with poor patient outcomes. Moreover, MPZL3 is necessary for homotypic cancer cell adhesion, and decreasing MPZL3 expression enhances invasion and clearance of mesothelial cell monolayers. In addition, MPZL3 loss abrogated cell cycle progression and proliferation. This was associated with increased resistance to Cisplatin and Olaparib and reduced DNA damage and apoptosis in response to these agents. Enhanced Cisplatin resistance was further validated in vivo. These data demonstrate for the first time that MPZL3 acts as an adhesion molecule and that MPZL3 loss results in EMT, decreased proliferation, and drug resistance in ovarian cancer. Our study suggests that decreased MPZL3 expression is a phenotype of ovarian cancer tumor progression and metastasis and may contribute to treatment failure in advanced-stage patients.

cancer biology↗

bioRxiv2024

Hypomorphic NOTCH1 Expression Alters Cardiomyocyte Cellular Architecture in Hypoplastic Left Heart Syndrome

NOTCH1 is a protein involved in cardiac development and mutations in NOTCH1 are implicated in left sided congenital heart disease, including hypoplastic left heart syndrome (HLHS). Therapeutic advances for HLHS patients have been hampered by the absence of suitable experimental models. Here, using human induced pluripotent stem cells (hiPSCs), we have generated a series of CRISPR-edited complex heterozygous hypomorphic mutations in NOTCH1 that recapitulate mutations seen in HLHS patients. These cells demonstrate the downregulation of genes associated with mitochondria, the actin cytoskeleton, and cardiomyocyte development. In addition, these hypomorphic NOTCH1 hiPSCs cells showed an increased propensity to differentiate towards non-cardiomyocyte lineages such as fibroblasts and smooth muscle cells, a finding confirmed in HLHS patient-derived myocardial tissue. Abnormalities in sarcomeric and mitochondrial architecture contributed to decreased ATP production, abnormal calcium transients, and reduced contractility. Using a split nano-luciferase system as a NOTCH1 intracellular reporter, we screened a library of FDA-approved compounds. Auranofin, an agent currently employed for rheumatoid arthritis, was identified as a candidate drug that could rescue impaired differentiation and contractility seen in the hypomorphic NOTCH1 model. These findings point to cell-autonomous abnormalities in hypomorphic NOTCH1 pre-cardiomyocyte cells that can be leveraged to identify potential therapeutic strategies for patients with severe congenital cardiac abnormalities. HighlightsO_LICRISPR edited and patient-derived iPSCs with hypomorphic NOTCH1 expression were used to characterize a cell-based model of hypoplastic left heart syndrome. C_LIO_LIHypomorphic NOTCH1 iPSCs have abnormalities in pathways associated with mitochondrial function, actin cytoskeleton, and cardiomyocyte development. C_LIO_LIHypomorphic NOTCH1 iPSCs have less pluripotency and tend to skew differentiation away from cardiomyocytes and towards fibroblasts and smooth muscle cells. C_LIO_LINOTCH1 is required for cardiac cytoskeletal and mitochondrial architecture, and to preserve contractility and ATP production. C_LIO_LIA high-throughput drug screen identified auranofin as an agent that may benefit patients with hypoplastic left heart syndrome. C_LI

cell biology↗

bioRxiv2024

PRDM16-DT is a Brain and Astrocyte-Specific lncRNA Implicated in Alzheimers Disease

Astrocytes provide crucial support for neurons, contributing to synaptogenesis, synaptic maintenance, and neurotransmitter recycling. Under pathological conditions, deregulation of astrocytes contributes to neurodegenerative diseases such as Alzheimers disease (AD), highlighting the growing interest in targeting astrocyte function to address early phases of AD pathogenesis. While most research in this field has focused on protein-coding genes, non-coding RNAs, particularly long non-coding RNAs (lncRNAs), have emerged as significant regulatory molecules. In this study, we identified the lncRNA PRDM16-DT as highly enriched in the human brain, where it is almost exclusively expressed in astrocytes. PRDM16-DT and its murine homolog, Prdm16os, are downregulated in the brains of AD patients and in AD models. In line with this, knockdown of PRDM16-DT and Prdm16os revealed its critical role in maintaining astrocyte homeostasis and supporting neuronal function by regulating genes essential for glutamate uptake, lactate release, and neuronal spine density through interactions with the RE1-Silencing Transcription factor (Rest) and Polycomb Repressive Complex 2 (PRC2). Notably, CRISPR-mediated overexpression of Prdm16os mitigated functional deficits in astrocytes induced by stimuli linked to AD pathogenesis. These findings underscore the importance of PRDM16-DT in astrocyte function and its potential as a novel therapeutic target for neurodegenerative disorders characterized by astrocyte dysfunction

neuroscience↗

bioRxiv2024

αKG-mediated carnitine synthesis promotes homologous recombination via histone acetylation

Homologous recombination (HR) deficiency enhances sensitivity to DNA damaging agents commonly used to treat cancer. In HR-proficient cancers, metabolic mechanisms driving response or resistance to DNA damaging agents remain unclear. Here we identified that depletion of alpha-ketoglutarate (KG) sensitizes HR-proficient cells to DNA damaging agents by metabolic regulation of histone acetylation. KG is required for the activity of KG-dependent dioxygenases (KGDDs), and prior work has shown that changes in KGDD affect demethylases. Using a targeted CRISPR knockout library consisting of 64 KGDDs, we discovered that Trimethyllysine Hydroxylase Epsilon (TMLHE), the first and rate-limiting enzyme in de novo carnitine synthesis, is necessary for proliferation of HR-proficient cells in the presence of DNA damaging agents. Unexpectedly, KG-mediated TMLHE-dependent carnitine synthesis was required for histone acetylation, while histone methylation was affected but dispensable. The increase in histone acetylation via KG-dependent carnitine synthesis promoted HR-mediated DNA repair through site- and substrate-specific histone acetylation. These data demonstrate for the first time that HR-proficiency is mediated through KG directly influencing histone acetylation via carnitine synthesis and provide a metabolic avenue to induce HR-deficiency and sensitivity to DNA damaging agents.

cancer biology↗

bioRxiv2024

GPX3 supports ovarian cancer tumor progression in vivo and promotes expression of GDF15

ObjectiveWe previously reported that high expression of the extracellular glutathione peroxidase GPX3 is associated with poor patient outcome in ovarian serous adenocarcinomas, and that GPX3 protects ovarian cancer cells from oxidative stress in culture. Here we tested if GPX3 is necessary for tumor establishment in vivo and to identify novel downstream mediators of GPX3s pro-tumorigenic function. MethodsGPX3 was knocked-down in ID8 ovarian cancer cells by shRNA to test the role of GPX3 in tumor establishment using a syngeneic IP xenograft model. RNA sequencing analysis was carried out in OVCAR3 cells following shRNA-mediated GPX3 knock-down to identify GPX3-dependent gene expression signatures. ResultsGPX3 knock-down abrogated clonogenicity and intraperitoneal tumor development in vivo, and the effects were dependent on the level of GPX3 knock-down. RNA sequencing showed that loss of GPX3 leads to decreased gene expression patterns related to pro-tumorigenic signaling pathways. Validation studies identified GDF15 as strongly dependent on GPX3. GDF15, a member of the TGF-{beta} growth factor family, has known oncogenic and immune modulatory activities. Similarly, GPX3 expression positively correlated with pro-tumor immune cell signatures, including regulatory T-cell and macrophage infiltration, and displayed significant correlation with PD-L1 expression. ConclusionsWe show for the first time that tumor produced GPX3 is necessary for ovarian cancer growth in vivo and that it regulates expression of GDF15. The immune profile associated with GPX3 expression in serous ovarian tumors suggests that GPX3 may be an alternate marker of ovarian tumors susceptible to immune check-point inhibitors.

cancer biology↗

bioRxiv2023

Development of adaptive anoikis resistance promotes metastasis that can be overcome by CDK8/19 Mediator kinase inhibition

Anoikis resistance or evasion of cell death triggered by matrix detachment is a hallmark of cancer cell survival and metastasis. We show that repeated exposure to suspension stress followed by recovery under attached conditions leads to development of anoikis resistance. The acquisition of anoikis resistance is associated with enhanced invasion, chemoresistance, and immune evasion in vitro and distant metastasis in vivo. This acquired anoikis resistance is not genetic, persisting for a finite duration without detachment stress, but is sensitive to CDK8/19 Mediator kinase inhibition that can also reverse anoikis resistance. Transcriptomic analysis reveals that CDK8/19 kinase inhibition induces bidirectional transcriptional changes in both sensitive and resistant cells, disrupting the balanced reprogramming required for anoikis adaptation and resistance by reversing some resistance associated pathways and enhancing others. Both anoikis resistance and in vivo metastatic growth of ovarian cancers are sensitive to CDK8/19 inhibition, thereby providing a therapeutic opportunity to both prevent and suppress ovarian cancer metastasis.

cancer biology↗

bioRxiv2023

Paracrine effects of the senescence-associated secretory phenotype decrease cancer cell adhesion

Cellular senescence, characterized by a stable cell cycle arrest, is a well-documented consequence of several widely used chemotherapeutics that has context-dependent roles in cancer. Although senescent cells are non-proliferative, they remain biologically active and secrete a complex and diverse array of factors collectively known as the se-nescence-associated secretome (SAS), which exerts pro-tumorigenic effects. Here, we aimed to mechanistically investigate how the SAS contributes to metastatic dissemination of high grade serous ovarian cancer (HGSOC) using standard-of-care cisplatin as a se-nescence inducer. Our findings demonstrate that the cisplatin-induced SAS enhances the dissemination of HGSOC in vivo without affecting cell proliferation or viability. We found that the SAS facilitates cell detachment, an effect that is mediated by a metabolic com-ponent. Using a metabolically focused CRISPR knockout screen, we identified complex I as the key driver of SAS-mediated cell detachment in bystander cells and validated that inhibition of complex I activity decreases HGSOC dissemination in vivo. Mechanistically, this effect was driven by SAS-mediated inhibition of an NAD+-SIRT-SREBP axis, leading to decreased plasma membrane cholesterol that increased cell detachment. Excitingly, we found that fructose is the key SAS component upstream of the NAD+-SIRT-SREBP-cholesterol axis mediating increased detachment of bystander cells, and a high fructose diet increases HGSOC dissemination in vivo. These findings reveal that the cisplatin-induced SAS reprograms the metabolic microenvironment in HGSOC, driving cancer cell detachment and promoting metastatic dissemination in a paracrine fashion. They also point to a previously unrecognized pro-tumorigenic effect of the SAS that may contribute to the high recurrence rate of HGSOC patients.

cancer biology↗

bioRxiv2023

ATR promotes mTORC1 activation via de novo cholesterol synthesis in p16-low cancer cells

DNA damage and cellular metabolism exhibit a complex interplay characterized by bidirectional feedback mechanisms. Key mediators of the DNA damage response and cellular metabolic regulation include Ataxia Telangiectasia and Rad3-related protein (ATR) and the mechanistic Target of Rapamycin Complex 1 (mTORC1), respectively. Previous studies have established ATR as a regulatory upstream factor of mTORC1 during replication stress; however, the precise mechanisms by which mTORC1 is activated in this context remain poorly defined. Additionally, the activity of this signaling axis in unperturbed cells has not been extensively investigated. Here, we demonstrate that ATR promotes mTORC1 activity across various cellular models under basal conditions. This effect is particularly enhanced in cells following the loss of p16, which we have previously associated with hyperactivation of mTORC1 signaling and here found have increased ATR activity. Mechanistically, we found that ATR promotes de novo cholesterol synthesis and mTORC1 activation through the upregulation of lanosterol synthase (LSS), independently of both CHK1 and the TSC complex. Furthermore, the attenuation of mTORC1 activity resulting from ATR inhibition was rescued by supplementation with lanosterol or cholesterol in multiple cellular contexts. This restoration corresponded with enhanced localization of mTOR to the lysosome. Collectively, our findings demonstrate a novel connection linking ATR and mTORC1 signaling through the modulation of cholesterol metabolism.

cancer biology↗

bioRxiv2023

Loss of STIM2 in colorectal cancer drives growth and metastasis through metabolic reprogramming and PERK-ATF4 endoplasmic reticulum stress pathway

The endoplasmic reticulum (ER) stores large amounts of calcium (Ca2+), and the controlled release of ER Ca2+ regulates a myriad of cellular functions. Although altered ER Ca2+ homeostasis is known to induce ER stress, the mechanisms by which ER Ca2+ imbalance activate ER stress pathways are poorly understood. Stromal-interacting molecules STIM1 and STIM2 are two structurally homologous ER-resident Ca2+ sensors that synergistically regulate Ca2+ influx into the cytosol through Orai Ca2+ channels for subsequent signaling to transcription and ER Ca2+ refilling. Here, we demonstrate that reduced STIM2, but not STIM1, in colorectal cancer (CRC) is associated with poor patient prognosis. Loss of STIM2 causes SERCA2-dependent increase in ER Ca2+, increased protein translation and transcriptional and metabolic rewiring supporting increased tumor size, invasion, and metastasis. Mechanistically, STIM2 loss activates cMyc and the PERK/ATF4 branch of ER stress in an Orai-independent manner. Therefore, STIM2 and PERK/ATF4 could be exploited for prognosis or in targeted therapies to inhibit CRC tumor growth and metastasis. HighlightsO_LISTIM2 regulates ER Ca2+ homeostasis independently of Orai and SOCE. C_LIO_LISTIM2 downregulation in colorectal cancer cells causes enhanced ER Ca2+ and is associated with poor patient prognosis. C_LIO_LISTIM2 downregulation induces PERK/ATF4 dependent ER stress in colorectal cancer. C_LIO_LIIncreased ER stress drives colorectal cancer metabolic reprogramming, growth, and metastasis. C_LI

cancer biology↗

bioRxiv2023

Alternate splice variants of the mitochondrial fission protein DNM1L/Drp1 regulate mitochondrial dynamics and cell fate in ovarian cancer.

Aberrant mitochondrial fission/fusion dynamics have been reported in cancer cells. While post translational modifications are known regulators of the mitochondrial fission/fusion machinery, we show that alternative splice variants of the fission protein Drp1 (DNM1L) have specific and unique roles in cancer, adding to the complexity of mitochondrial fission/fusion regulation in tumor cells. Ovarian cancer specimens express an alternative splice transcript variant of Drp1 lacking exon 16 of the variable domain, and high expression of this splice variant relative to other transcripts is associated with poor patient outcome. Unlike the full-length variant, expression of Drp1 lacking exon 16 leads to decreased association of Drp1 to mitochondrial fission sites, more fused mitochondrial networks, enhanced respiration, and TCA cycle metabolites, and is associated with a more metastatic phenotype in vitro and in vivo. These pro-tumorigenic effects can also be inhibited by specific siRNA-mediated inhibition of the endogenously expressed transcript lacking exon 16. Moreover, lack of exon 16 abrogates mitochondrial fission in response to pro-apoptotic stimuli and leads to decreased sensitivity to chemotherapeutics. These data emphasize the significance of the pathophysiological consequences of Drp1 alternative splicing and divergent functions of Drp1 splice variants, and strongly warrant consideration of Drp1 splicing in future studies.

cancer biology↗

bioRxiv2023

Heparan sulfate modifications of betaglycan promote TIMP3-dependent ectodomain shedding to fine-tune TGF-β signaling.

In pathologies such as cancer, aberrant Transforming Growth Factor-{beta} (TGF-{beta}) signaling exerts profound tumor intrinsic and extrinsic consequences. Intense clinical endeavors are underway to target this pivotal pathway. Central to the success of these interventions is pinpointing factors that decisively modulate the TGF-{beta} responses. Betaglycan/type III TGF-{beta} receptor (T{beta}RIII), is an established co-receptor for the TGF-{beta} superfamily known to bind directly to TGF-{beta}s 1-3 and inhibin A/B. While betaglycan can be membrane-bound, it can also undergo ectodomain cleavage to produce soluble-betaglycan that can sequester its ligands. The extracellular domain of betaglycan undergoes heparan sulfate and chondroitin sulfate glycosaminoglycan modifications, transforming betaglycan into a proteoglycan. Here we report the unexpected discovery that the heparan sulfate modifications are critical for the ectodomain shedding of betaglycan. In the absence of such modifications, betaglycan is not shed. Such shedding is indispensable for the ability of betaglycan to suppress TGF-{beta} signaling and the cells responses to exogenous TGF-{beta} ligands. Using unbiased transcriptomics, we identified TIMP3 as a key regulator of betaglycan shedding and thereby TGF-{beta} signaling. Our results bear significant clinical relevance as modified betaglycan is present in the ascites of patients with ovarian cancer and can serve as a marker for predicting patient outcomes and TGF-{beta} signaling responses. These studies are the first to demonstrate a unique reliance on the glycosaminoglycan modifications of betaglycan for shedding and influence on TGF-{beta} signaling responses. Dysregulated shedding of TGF-{beta} receptors plays a vital role in determining the response and availability of TGF-{beta}s, which is crucial for prognostic predictions and understanding of TGF-{beta} signaling dynamics.

biochemistry↗

bioRxiv2022

Carcinoma associated mesenchymal stem cells promote ovarian cancer metastasis by increasing tumor heterogeneity through direct mitochondrial transfer

Ovarian cancer is characterized by early, diffuse metastatic spread with most women presenting with extensive abdominal metastasis at the time of diagnosis. Prior work demonstrated carcinoma-associated mesenchymal stem cells (CA-MSCs) enhance ovarian cancer metastasis through a process of direct cellular interaction and formation of heterocellular CA-MSC and tumor cell complexes. In this study, we demonstrated that CA-MSCs enhance metastasis by increasing tumor cell heterogeneity through mitochondrial donation. We showed that CA-MSCs directly interacted with ovarian cancer cells via tunneling nanotubules (TNTs), and CA-MSCs used these TNTs to transfer live mitochondria to adjacent ovarian cancer cells. This mitochondrial donation preferentially occurred with ovarian cancer cells that had the lowest mitochondrial mass, as quantified using live, actively respiring mitochondrial labeling. These mito poor cancer cells demonstrated decreased proliferation, increased sensitivity to chemotherapy, and decreased oxidative phosphorylation compared to mito rich cancer cells. CA-MSCs rescued the phenotypes of mito poor cancer cells, restoring their proliferative capacity, increasing chemotherapy resistance, and increasing oxidative phosphorylation. We validated these findings in a fully autologous system using CA-MSCs and cancer cells derived from the same patient to prevent confounding effects of cellular response to foreign organelle/DNA. Using a knockdown of the mitochondrial motor protein, MIRO1, in CA-MSCs, we demonstrated that mitochondrial transfer is necessary for the CA-MSC-mediated rescue of mito poor cancer cells. Mitochondria of CA-MSC origin persisted in tumor cells over multiple passages. Importantly, CA-MSC mitochondrial donation occurred in vivo, significantly enhanced tumor cell heterogeneity and decreased survival in an orthotopic ovarian cancer mouse model. Collectively, this work identified CA-MSC mitochondrial transfer as a critical mediator of ovarian cancer cell survival, heterogeneity, and metastasis, and blocking CA-MSC mitochondrial transfer represents a unique therapeutic target in ovarian cancer.

cancer biology↗

bioRxiv2022

PVT1, a YAP1 dependent stress responsive lncRNA drives ovarian cancer metastasis and chemoresistance

Metastatic growth of ovarian cancer cells into the peritoneal cavity requires adaptation to various cellular stress factors to facilitate cell survival and growth. Here we demonstrate the role of PVT1, one such stress induced long non-coding RNA, in ovarian cancer growth and metastasis. PVT1 is an amplified and overexpressed lncRNA in ovarian cancer with strong predictive value for survival and response to targeted therapeutics. We find that expression of PVT1 is regulated by ovarian tumor cells in response to cellular stress, particularly loss of cell-cell contacts and changes in matrix rigidity occurring in a YAP1 dependent manner. Induction of PVT1 promotes tumor cell survival, growth, and migration. Conversely, reducing PVT1 levels robustly abrogates metastatic behavior and tumor cell dissemination in cell lines and syngeneic transplantation models in vivo. We find that reducing PVT1 causes widespread transcriptome changes leading to alterations in cellular stress response and metabolic pathways including doxorubicin metabolism, which directly impacts chemosensitivity. Together, these findings implicate PVT1 as a promising therapeutic target to suppress metastasis and avoid chemoresistance in ovarian cancer. SUMMARY BLURBPVT1 is a stress-responsive IncRNA regulated by YAP1, leading to increased ovarian cancer metastasis and chemoresistance, making PVT1 a promising therapeutic target.

cancer biology↗

bioRxiv2022

Reciprocal epigenetic Sox2 regulation by SMAD1-SMAD3 is critical for anoikis resistance and metastasis in cancer

Growth factors in the tumor environment are key regulators of cell survival and metastasis. Here we reveal, dichotomy between TGF-{beta} superfamily growth factors BMP and TGF-{beta}/activin, and their downstream SMAD effectors. Gene expression profiling uncovered Sox2 as a key signaling node regulated in an opposing manner by anoikis-promoting BMP2, 4 and 9, and anoikis-suppressing TGF-{beta} and activin A. We find that Sox2 repression by BMPs robustly inhibits intraperitoneal tumor burden and increases survival in multiple ovarian cancer models. Repression of Sox2 is driven by SMAD1 dependent histone H3K27me3 recruitment and DNA methylation at SOX2s promoter. Conversely, TGF-{beta} and activin A promote Sox2 expression, and anoikis resistance by SMAD3 mediated histone H3K4me3 recruitment. We find that balancing Sox2 levels is critical for anoikis, as transcriptomics reveals regulation of key cell death pathways. Moreover, BMP-driven SMAD1 signaling can override TGF-{beta} and activins effect on Sox2, which has clinical significance due to the high levels of TGF-{beta} we find in ovarian cancer patients. Together, our findings identify Sox2 as a contextual and contrastingly regulated key node, downstream of TGF-{beta} superfamily members controlling anoikis and metastasis in ovarian cancers. HighlightsO_LISox2 is a key node for anoikis resistance in cancer C_LIO_LISox2 is differentially regulated by TGF-{beta}/activin and BMPs in broad cancers C_LIO_LIBMP9 is a robust metastasis suppressor by lowering Sox2 C_LIO_LISox2 regulation is contextual, epigenetic and at the transcriptional level C_LI

cancer biology↗