Pro-survival Role Research Articles

The Mitochondrial Ubiquitin Ligase MARCHF5 Cooperates with MCL1 to Inhibit Apoptosis in KSHV-Transformed Primary Effusion Lymphoma Cell Lines.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several malignancies in people with HIV including Kaposi's sarcoma and primary effusion lymphoma (PEL). We have previously shown that PEL cell lines require myeloid cell leukemia-1 (MCL1) to inhibit apoptosis. MCL1 is an oncogene that is amplified in cancers and causes resistance to chemotherapy regimens. MCL1 is thus an attractive target for drug development. The emerging clinical relevance and therapeutic potential of MCL1 motivated us to study the roles of this oncogene in PEL in depth. Using a systems biology approach, we uncovered an unexpected genetic interaction between MCL1 and MARCHF5 indicating that they function in the same pathway. MARCHF5 is an E3 ubiquitin ligase most known for regulating mitochondrial homeostasis and antiviral signaling, but not apoptosis. We thus investigated how MCL1 and MARCHF5 cooperate to promote PEL cell survival. CRISPR knockout (KO) of MARCHF5 in PEL cell lines resulted in a significant increase in apoptosis despite the presence of MCL1. The anti-apoptotic function of MARCHF5 was dependent on its E3 ligase and dimerization activities. Loss of MARCHF5 or inhibition of the 26S proteasome furthermore stabilized the MCL1 antagonist NOXA without affecting levels of MCL1. Interestingly, NOXA KO provides a fitness advantage to PEL cells suggesting that NOXA is the pro-apoptotic signal that necessitates the anti-apoptotic activities of MCL1 and MARCHF5. Finally, endogenous reciprocal co-immunoprecipitation experiments show that MARCHF5 and NOXA are found in the same protein complex. Our findings thus provide the mechanistic link that underlies the genetic interaction between MCL1 and MARCHF5. We propose that MARCHF5 induces the degradation of the MCL1 antagonist NOXA thus reinforcing the pro-survival role of MCL1 in these tumor cells. This newly appreciated interaction of the MCL1 and MARCHF5 oncogenes may be useful to improve the design of combination therapies for KSHV malignancies.

Neuregulin 1 Signaling Attenuates Tumor Necrosis Factor α-Induced Female Rat Luteal Cell Death.

The corpus luteum (CL) is a transient ovarian endocrine structure that maintains pregnancy in primates during the first trimester and in rodents during the entire pregnancy by producing steroid hormone progesterone (P4). CL lifespan, growth, and differentiation are tightly regulated by survival and cell death signals through luteotrophic and luteolytic factors, including the epidermal growth factor (EGF)-like factor family. Neuregulin 1 (NRG1), a member of the EGF family, mediates its effect through ErbB2/3 receptors. However, the functional role of NRG1 in luteal cells (LCs) is unknown. Thus, this study investigated the role of NRG1 and its molecular mechanism of action in rat LC. Our experimental results suggest a strong positive correlation between steroidogenic acute regulatory protein (StAR) and NRG1 expression in mid-CL and serum P4 and estrogen (E2) production. In contrast, there was a decrease in StAR and NRG1 expression and P4 and E2 production with an increase in tumor necrosis factor α (TNFα) expression in regressing CL. Further in vitro studies in LCs showed that the knockdown of endogenous Nrg1 promoted the expression of proinflammatory and proapoptotic factors and decreased prosurvival factor expression. Subsequently, treatment with exogenous TNFα under these experimental conditions profoundly elevated proinflammatory and proapoptotic factors. Further analysis demonstrated that the phosphorylation status of ErbB2/3, PI3K, Ak strain transforming or protein kinase B (Akt), and ErK1/2 was significantly inhibited under these experimental conditions, whereas the treatment of TNFα further inhibited the phosphorylation of ErbB2/3, PI3K, Akt, and ErK1/2. Collectively, these studies provide new insights into the NRG1-mediated immunomodulatory and prosurvival role in LCs, which may maintain the function of CL.

Secreted GDF15 maintains transcriptional responses during DNA damage-mediated senescence in human beta cells.

Type 1 diabetes (T1D) is a chronic metabolic disease resulting from an autoimmune destruction of pancreatic beta cells. Beta cells activate various stress responses during the development of T1D, including senescence, which involves cell cycle arrest, prosurvival signaling, and a proinflammatory secretome termed the senescence-associated secretory phenotype (SASP). We previously identified growth and differentiation factor 15 (GDF15) as a major SASP factor in human islets and human EndoC-βH5 beta cells in a model of DNA damage-mediated senescence that recapitulates features of senescent beta cells in T1D. Soluble GDF15 has been shown to exert protective effects on human and mouse beta cells during various forms of stress relevant to T1D; therefore, we hypothesized that secreted GDF15 may play a prosurvival role during DNA damage-mediated senescence in human beta cells. We found that elevated GDF15 secretion was associated with endogenous senescent beta cells in an islet preparation from a T1D donor, supporting the validity of our DNA damage model. Using antibody-based neutralization, we found that secreted endogenous GDF15 was not required for senescent human islet or EndoC cell viability. Rather, neutralization of GDF15 led to reduced expression of specific senescence-associated genes, including GDF15 itself and the prosurvival gene BCL2-like protein 1 (BCL2L1). Taken together, these data suggest that SASP factor GDF15 is not required to sustain senescent human islet viability, but it is required to maintain senescence-associated transcriptional responses.NEW & NOTEWORTHY Beta cell senescence is an emerging contributor to the pathogenesis of type 1 diabetes, but candidate therapeutic targets have not been identified in human beta cells. In this study, we examined the role of a secreted factor, GDF15, and found that although it is not required to maintain viability during senescence, it is required to fine-tune gene expression programs involved in the senescence response during DNA damage in human beta cells.

MANF facilitates breast cancer cell survival under glucose-starvation conditions via prkn-mediated mitophagy regulation

ABSTRACT During tumor expansion, breast cancer (BC) cells often experience reactive oxygen species accumulation and mitochondrial damage because of glucose shortage. However, the mechanism by which BC cells deal with the glucose-shortage-induced oxidative stress remains unclear. Here, we showed that MANF (mesencephalic astrocyte derived neurotrophic factor)-mediated mitophagy facilitates BC cell survival under glucose-starvation conditions. MANF-mediated mitophagy also promotes fatty acid oxidation in glucose-starved BC cells. Moreover, during glucose starvation, SENP1-mediated de-SUMOylation of MANF increases cytoplasmic MANF expression through the inhibition of MANF’s nuclear translocation and hence renders mitochondrial distribution of MANF. MANF mediates mitophagy by binding to PRKN (parkin RBR E3 ubiquitin protein ligase), a key mitophagy regulator, in the mitochondria. Under conditions of glucose starvation, protein oxidation inhibits PRKN activity; nevertheless, the CXXC motif of MANF alleviates protein oxidation in RING II-domain of PRKN and restores its E3 ligase activity. Furthermore, MANF-PRKN interactions are essential for BC tumor growth and metastasis. High MANF expression predicts poor outcomes in patients with BC. Our results highlight the prosurvival role of MANF-mediated mitophagy in BC cells during glucose starvation, suggesting MANF as a potential therapeutic target.

BAG3 Mediated Down-regulation in Expression of p66shc has Ramifications on Cellular Proliferation, Apoptosis and Metastasis.

Redundancy of cancer cells towards ROS-mediated apoptosis despite expressing proline-rich p66shc abundantly needs to be investigated properly. P66shc, an adapter protein, is indispensable both for initiating ROS-mediated apoptosis and subsequent ROS generation through Rac-1 activation. P66shc gets phosphorylated at Ser-36 that triggers its translocation to the mitochondria and subsequent release of Cytochrome c in response to oxidative stress. It also aids in Rac-1 dependent NADPH oxidase activation, leading to the generation of cytosolic ROS that can perform diverse functions depending on its concentration. This study has identified the multi-faceted anti-apoptotic protein BAG3 as an interacting partner of p66shc. BAG3 utilizes its WW domain to bind to the proline-rich motifs of p66shc. BAG3, through its WW domain, antagonizes p66shc mediated apoptosis, by inhibiting both the expression and phosphorylation of p66shc under normal and oxidative stress conditions. This results in significant protection against ROS-mediated apoptosis. BAG3-mediated reduction in p66shc expression increases cell proliferation and metastasis. The increase in cell proliferation is attributed to the impact of BAG3 on Rac-1 activation and ROS production under normal conditions. This study has unraveled an interactor of p66shc that enhances pro-survival role while simultaneously suppressing its apoptotic role.

HDAC3 genetic and pharmacologic inhibition radiosensitizes fusion positive rhabdomyosarcoma by promoting DNA double-strand breaks

Radiotherapy (RT) plays a critical role in the management of rhabdomyosarcoma (RMS), the prevalent soft tissue sarcoma in childhood. The high risk PAX3-FOXO1 fusion-positive subtype (FP-RMS) is often resistant to RT. We have recently demonstrated that inhibition of class-I histone deacetylases (HDACs) radiosensitizes FP-RMS both in vitro and in vivo. However, HDAC inhibitors exhibited limited success on solid tumors in human clinical trials, at least in part due to the presence of off-target effects. Hence, identifying specific HDAC isoforms that can be targeted to radiosensitize FP-RMS is imperative. We, here, found that only HDAC3 silencing, among all class-I HDACs screened by siRNA, radiosensitizes FP-RMS cells by inhibiting colony formation. Thus, we dissected the effects of HDAC3 depletion using CRISPR/Cas9-dependent HDAC3 knock-out (KO) in FP-RMS cells, which resulted in Endoplasmatic Reticulum Stress activation, ERK inactivation, PARP1- and caspase-dependent apoptosis and reduced stemness when combined with irradiation compared to single treatments. HDAC3 loss-of-function increased DNA damage in irradiated cells augmenting H2AX phosphorylation and DNA double-strand breaks (DSBs) and counteracting irradiation-dependent activation of ATM and DNA-Pkcs as well as Rad51 protein induction. Moreover, HDAC3 depletion hampers FP-RMS tumor growth in vivo and maximally inhibits the growth of irradiated tumors compared to single approaches. We, then, developed a new HDAC3 inhibitor, MC4448, which showed specific cell anti-tumor effects and mirrors the radiosensitizing effects of HDAC3 depletion in vitro synergizing with ERKs inhibition. Overall, our findings dissect the pro-survival role of HDAC3 in FP-RMS and suggest HDAC3 genetic or pharmacologic inhibition as a new promising strategy to overcome radioresistance in this tumor.

MRNIP limits ssDNA gaps during replication stress.

Replication repriming by the specialized primase-polymerase PRIMPOL ensures the continuity of DNA synthesis during replication stress. PRIMPOL activity generates residual post-replicative single-stranded nascent DNA gaps, which are linked with mutagenesis and chemosensitivity in BRCA1/2-deficient models, and which are suppressed by replication fork reversal mediated by the DNA translocases SMARCAL1 and ZRANB3. Here, we report that the MRE11 regulator MRNIP limits the prevalence of PRIMPOL and MRE11-dependent ssDNA gaps in cells in which fork reversal is perturbed either by treatment with the PARP inhibitor Olaparib, or by depletion of SMARCAL1 or ZRANB3. MRNIP-deficient cells are sensitive to PARP inhibition and accumulate PRIMPOL-dependent DNA damage, supportive of a pro-survival role for MRNIP linked to the regulation of gap prevalence. In MRNIP-deficient cells, post-replicative gap filling is driven in S-phase by UBC13-mediated template switching involving REV1 and the TLS polymerase Pol-ζ. Our findings represent the first report of modulation of post-replicative ssDNA gap dynamics by a direct MRE11 regulator.

Rapid P-TEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy, opening avenues for combinatorial treatment in these lethal malignancies.

Antitumor effects of co-treatment of resveratrol with antitumor drugs in ER- and HER2-positive breast cancer cells are due to induction of apoptosis and modulation of estrogen receptor expression.

Resveratrol, a natural compound, may be an alternative to improving conventional breast cancer therapy. Thus, we assessed the capability of resveratrol at a low dose to enhance the in vitro effect of conventional theray in estrogen receptor (ER) and human epidermal growth factor receptor type 2 (HER2)-positive breast cancer cells. Cell viability of breast cancer cells was measured with neutral red uptake assay. Apoptosis, autophagy, cell cycle progression and cell proliferation were detected through hypotonic fluorescent solution assay, formation of acidic vesicular organelles, flow cytometry, and bromodeoxyuridine assay, respectively. Western blotting was performed to study the expression of pro-apoptotic, anti-apoptotic and autophagic proteins, and estrogen receptors. Resveratrol combined with tamoxifen metabolites or trastuzumab reduced cell viability of ER- and HER2-positive breast cancer cells, respectively. This effect was mainly associated with induction of apoptosis due to a greater formation of hypodiploid nuclei, reduced protein expression of procaspase-7, Bcl-2, Bcl-xL, and PARP; and increased expression of cleaved PARP. Resveratrol decreased the expression of ERα and increased that of ERβ, contributing to the reduced viability on breast cancer cells. Combined treatments induced autophagy, evidenced by increased levels of acidic vesicular organelles and degradation of p62/SQSTM1 protein. Nevertheless, on inhibiting autophagy with 3-methyladenine, cell viability was further reduced and apoptosis was induced, suggesting a pro-survival role of autophagy, impairing apoptosis. Resveratrol increasead the in vitro cytotoxic effect of conventional therapy in breast cancer cells. However, it was necessary to block resveratrol-induced autophagy to improve the therapeutic response.

Abstract 5996: Genome-wide CRISPR screening reveals a central role for ferroptotic cell death in the anti-tumor response to mTOR inhibitors in HNSCC

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer globally, resulting in more than 300,000 deaths each year worldwide. Treatment options for HNSCC patients include surgery, radiation, chemotherapy and molecularly targeted therapies, and although immunotherapies have recently revolutionized the treatment landscape, <20% of HNSCC patients respond to immune check point blockade (ICB) therapies. The survival rate of HNSCC patients has changed only modestly over the past decades, and therefore HNSCC is a significant global health problem with high mortality and morbidity. Genomic alterations converging in the persistent activation of the PI3K/mTOR pathway (>80% cases) represent one of the most frequently altered signaling circuitries in HNSCC. This overreliance on PI3K/mTOR signaling for tumor growth may expose a cancer vulnerability that can be exploited therapeutically, as revealed in recent clinical trials using mTOR inhibitors (mTORi) in HNSCC in the adjuvant and neoadjuvant setting. Here, we took advantage of a whole-genome CRISPR screening approach to identify mechanisms of sensitivity and resistance to mTORi, aimed at increasing their clinical activity. We found that the activation of autophagy, a biological process that plays a paradoxical pro-survival or antitumoral role in a cancer specific fashion, is strictly required for mTORi cell growth inhibition in HNSCC. In depth analysis of our CRISPR screening also revealed multiple hits suggestive of a novel role for iron metabolism and ferroptosis downstream from mTORi. Ferroptosis is iron-dependent regulated cell death process caused by the peroxidation of polyunsaturated fatty acids. Indeed, we found that mTORi induces ferroptosis in HNSCC cells, and that inhibition of ferroptosis reduces the effect of mTORi. Evidence will be presented supporting a novel mechanism linking mTORi-triggered autophagy and the activation of ferroptotic death programs, as well as new synergistic combinations with mTORi by repurposing approved drugs that disable cellular ferroptotic defense mechanisms. Our studies uncovered how mTORi act in HNSCC, thereby revealing new multimodal precision therapies for HNSCC and many human malignancies displaying overactive PI3K/mTOR signaling. Citation Format: Keiichi Koshizuka, Xingyu Wu, Kuniaki Sato, Pham Thuy Vo, Gosia M. Murawska, Tomohiko Ishikawa, Zhiyong Wang, Alfredo A. Molinolo, Edward A. Dennis, Prashant Mali, J. Silvio Gutkind. Genome-wide CRISPR screening reveals a central role for ferroptotic cell death in the anti-tumor response to mTOR inhibitors in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5996.

Abstract 5357: The tumor-intrinsic RNA binding protein HuR is essential for anti-tumor immunity in PDAC

Abstract Immunotherapies have shown limited effectiveness in pancreatic ductal adenocarcinoma (PDAC), despite having success in other cancers. The RNA-binding protein HuR is known to play a critical role in the oncogenesis of PDAC by regulating key mRNA transcripts. Our lab has shown that tumor-intrinsic HuR is overexpressed in PDAC and regulates the tumor microenvironment composition, altering multiple tumor cytokines to release. Thus, we hypothesized that HuR plays a role in immune evasion of PDAC. We disrupted the locus encoding HuR (Elavl1) using CRISPR Cas9 in Kras-p53 mutant-driven (KPC) murine PDAC to assess its function in tumor immune evasion. Here, we report that HuR, a known pro-survival factor for PDAC, has a role in tumor immune evasion by suppressing T cell infiltration and T cell activation. Specifically, HuR-knockout (KO) KPC tumors grew slower compared to KPC wildtype (WT) tumors in an orthotopically implanted model (p-value < 0.0001), even though human PDAC cell lines with HuR-WT and HuR-KO grew at the same rate in NRG mice (in an immune-compromised environment). Accordingly, HuR-KO tumors had more T cell infiltration and activated T cells compared to HuR-WT tumors (p-value = 0.0047). Importantly, T cell depletion (both CD4+ and CD8+) partially rescued the size of HuR-KO tumors. Next, we rescued the expression of HuR in the HuR-KO cell line, and the re-expression partially rescued the tumor size in vivo. This observation validates a HuR-mediated dependent mechanism. Collectively, these data support the hypothesis that HuR suppresses T cell activation and prevents T cells from infiltrating into the PDAC microenvironment, leading to PDAC immune evasion. Ongoing experiments will transduce KPC cell lines with ovalbumin (OVA) antigen, which will selectively activate CD8+ OT-I T cells through their transgenic TCR, to measure the ability of KPC HuR-WT or HuR-KO OVA cells in directly activating CD8+ OT-I T cells. We will also investigate HuR's impact on immune cells' cytokine release and how that will impact the cytotoxicity of T cells. These findings support the ongoing concept that HuR plays an important pro-survival role in PDAC in vivo, and targeting strategies against HuR being developed (e.g., siHuR nano-therapies and small molecule inhibitors) could potentially enhance PDAC sensitivity to immune-based anti-cancer therapies, such as checkpoint blockade and T cell transfer. Citation Format: Yifei Guo, Jennifer M. Finan, Alexandra Q. Bartlett, Jonathan R. Brody, Robert Eil. The tumor-intrinsic RNA binding protein HuR is essential for anti-tumor immunity in PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5357.

When Therapy-Induced Cancer Cell Apoptosis Fuels Tumor Relapse

Most therapeutic strategies for solid tumor malignancies are designed based on the hypothesis that cancer cells evade apoptosis to exhibit therapy resistance. This is somewhat surprising given that clinical studies published since the 1990s have demonstrated that increased apoptosis in solid tumors is associated with cancer aggressiveness and poor clinical outcome. This is consistent with more recent reports demonstrating non-canonical (pro-survival) roles for apoptotic caspases, including caspase 3, as well as the ability of cancer cells to recover from late stages of apoptosis via a process called anastasis. These activities are essential for the normal development and maintenance of a healthy organism, but they also enable malignant cells (including cancer stem cells) to resist anticancer treatment and potentially contribute to clinical dormancy (minimal residual disease). Like apoptosis, therapy-induced cancer cell dormancy (durable proliferation arrest reflecting various manifestations of genome chaos) is also not obligatorily a permanent cell fate. However, as briefly discussed herein, compelling pre-clinical studies suggest that (reversible) dormancy might be the “lesser evil” compared to treacherous apoptosis.

β2-Adrenoceptor Activation Favor Acquisition of Tumorigenic Properties in Non-Tumorigenic MCF-10A Breast Epithelial Cells.

Noradrenaline and adrenaline, and their cognate receptors, are currently accepted to participate in cancer progression. They may also participate in cancer initiation, although their role in this phase is much less explored. The aim of this work was to study the influence of adrenergic stimulation in several processes related to breast cancer carcinogenesis, using several adrenergic agonists in the MCF-10A non-tumorigenic breast cells. Activation of the β-adrenoceptors promoted an epithelial phenotype in MCF-10A cells, revealed by an increased expression of the epithelial marker E-cadherin and a decrease in the mesenchymal markers, N-cadherin and vimentin. MCF-10A cell motility and migration were also impaired after the β-adrenoceptors activation. Concomitant with this effect, β-adrenoceptors decrease cell protrusions (lamellipodia and filopodia) while increasing cell adhesion. Activation of the β-adrenoceptors also decreases MCF-10A cell proliferation. When the MCF-10A cells were cultured under low attachment conditions, activation the of β- (likely β2) or of α2-adrenoceptors had protective effects against cell death, suggesting a pro-survival role of these adrenoceptors. Overall, our results showed that, in breast cells, adrenoceptor activation (mainly through β-adrenoceptors) may be a risk factor in breast cancer by inducing some cancer hallmarks, providing a mechanistic explanation for the increase in breast cancer incidences that may be associated with conditions that cause massive adrenergic stimulation, such as stress.

Abstract A043: The tumor-intrinsic RNA binding protein HuR is essential for anti-tumor immunity in PDAC

Abstract Immunotherapies have shown limited effectiveness in pancreatic ductal adenocarcinoma (PDAC), despite successes in many other cancers. The RNA-binding protein HuR is known to play a critical role in the oncogenesis of PDAC through regulating key mRNA transcripts. Our lab has showed that tumor-intrinsic HuR is overexpressed in PDAC and regulates the tumor microenvironment composition and alters multiple tumor cytokine release. Thus, we hypothesized that HuR plays a role in immune evasion of PDAC. We disrupted the locus encoding HuR (Elavl1), using CRISPR Cas9 in Kras-p53 mutant-driven (KPC) murine PDAC to assess its function in tumor immune evasion. Here, we report that HuR, a known pro-survival factor for PDAC, has a role in tumor immune evasion by suppressing T cell infiltration and T cell activation. Specifically, HuR-knockout (KO) KPC tumors grew slower compared to KPC wildtype (WT) tumors in an orthotopically implantation model (p-value<0.0001), even though they grew at the same rate in vitro (in a non-compromised environment). Accordingly, HuR-KO tumors had more T cell infiltration and activated T cells comparing to HuR-WT tumors (p-value=0.0047). Furthermore, T cell depletion partially rescued the size of HuR-KO tumors. Collectively, these data support the hypothesis that HuR suppresses T cell activation and prevents T cells from infiltrating the PDAC microenvironment, which therefore leads to PDAC immune evasion. Ongoing experiments will transduce KPC cell lines with ovalbumin (OVA) antigen, which will selectively active CD8+ OT-I T cells through their transgenic TCR, to measure the ability of KPC HuR-WT or HuR-KO OVA cells in directly activating CD8+ OT-I T cells. We will also rescue HuR in HuR-KO cells to validate HuR’s role in immune evasion. These findings support the concept that HuR plays an important pro-survival role of PDAC in vivo, and that targeting strategies against HuR being developed (e.g., siHuR nanotherapies and small molecule inhibitors) could potentially enhance PDAC sensitivity to immune-based anti-cancer therapies such as checkpoint blockade and T cell transfer. Citation Format: Yifei Guo, Jennifer M. Finan, Madeline D. Hedberg, Jonathan R. Brody, Robert Eil. The tumor-intrinsic RNA binding protein HuR is essential for anti-tumor immunity in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A043.

PD-L1 blockade TAM-dependently potentiates mild photothermal therapy against triple-negative breast cancer

The present work was an endeavor to shed light on how mild photothermia possibly synergizes with immune checkpoint inhibition for tumor therapy. We established mild photothermal heating protocols to generate temperatures of 43 °C and 45 °C in both in vitro and in vivo mouse 4T1 triple-negative breast cancer (TNBC) models using polyglycerol-coated carbon nanohorns (CNH-PG) and 808 nm laser irradiation. Next, we found that 1) CNH-PG-mediated mild photothermia (CNH-PG-mPT) significantly increased expression of the immune checkpoint PD-L1 and type-1 macrophage (M1) markers in the TNBC tumors; 2) CNH-PG-mPT had a lower level of anti-tumor efficacy which was markedly potentiated by BMS-1, a PD-L1 blocker. These observations prompted us to explore the synergetic mechanisms of CNH-PG-mPT and BMS-1 in the context of tumor cell-macrophage interactions mediated by PD-L1 since tumor-associated macrophages (TAMs) are a major source of PD-L1 expression in tumors. In vitro, the study then identified two dimensions where BMS-1 potentiated CNH-PG-mPT. First, CNH-PG-mPT induced PD-L1 upregulation in the tumor cells and showed a low level of cytotoxicity which was potentiated by BMS-1. Second, CNH-PG-mPT skewed TAMs towards an M1-like anti-tumor phenotype with upregulated PD-L1, and BMS-1 bolstered the M1-like phenotype. The synergistic effects of BMS-1 and CNH-PG-mPT both on the tumor cells and TAMs were more pronounced when the two cell populations were in co-culture. Further in vivo study confirmed PD-L1 upregulation both in tumor cells and TAMs in the TNBC tumors following treatment of CNH-PG-mPT. Significantly, TAMs depletion largely abolished the anti-TNBC efficacy of CNH-PG-mPT alone and in synergy with BMS-1. Collectively, our findings reveal PD-L1 upregulation to be a key response of TNBC to mild photothermal stress, which plays a pro-survival role in the tumor cells while also acting as a brake on the M1-like activation of the TAMs. Blockade of mPT‑induced PD‑L1 achieves synergistic anti-TNBC efficacy by taking the intrinsic survival edge off the tumor cells on one hand and taking the brakes off the M1-like TAMs on the other. Our findings reveal a novel way (i.e. mild thermia plus PD-L1 blockade) to modulate the TAMs-tumor cell interaction to instigate a mutiny of the TAMs against their host tumor cells.Graphical

YF343, A Novel Histone Deacetylase Inhibitor, Combined with CQ to Inhibit- Autophagy, Contributes to Increased Apoptosis in Triple- Negative Breast Cancer.

Compounds that target tumor epigenetic events are likely to constitute a prominent strategy for anticancer treatment. Histone deacetylase inhibitors (HDACis) have been developed as prospective candidates in anticancer drug development, and currently, many of them are under clinical investigation. We assessed the anticancer efficacy of a now hydroxamate-based HDACi, YF-343, in triple-negative breast cancer development and studied its potential mechanisms. YF-343 was estimated as a novel HDACi by the HDACi drug screening kit. The biological effects of YF-343 in a panel of breast cancer cell lines were analyzed by Western blot and flow cytometry. YF-343 exhibited notable cytotoxicity, promoted apoptosis, and induced cell cycle arrest. Furthermore, it also induced autophagy, which plays a pro-survival role in breast cancer cells. The combination of YF-343 with an autophagy inhibitor chloroquine (CQ) significantly suppressed breast tumor progression as compared to the YF-343 treatment alone both in vitro and in vivo. Mechanistically, the molecular mechanism of YF-343 on autophagy was elucidated by gene chip expression profiles, qPCR analysis, luciferase reporter gene assay, chromatin immunoprecipitation assays, immunohistochemical analysis, and other methods. E2F7, a transcription factor, promoted the expression of ATG2A via binding to the ATG2A promoter region and then induced autophagy in triple-negative breast cancer cells treated with YF-343. Our studies have illustrated the mechanisms for potential action of YF-343 on tumor growth in breast cancer models with pro-survival autophagy. The combination therapy of YF-343 and CQ maybe a promising strategy for breast cancer therapy.

FRI394 Anti-inflammatory Protein Neuregulin-1 (NRG1) And ErbB3 Receptor In Human Antral Follicle And Endometrium

Abstract Disclosure: I. Chowdhury: None. S. Banerjee: None. A. Rodriguez: None. Neuregulin 1 (NRG1) is a member of the EGF-like factor family and has recognized pro-survival and anti-inflammatory roles in neurodevelopmental and cardiac function, neurological and psychiatric disorders, cancer, and development. However, the physiological role of NRG1 in the human ovary and uterus has not been well documented. Therefore, we assessed the expression pattern of NRG1 and its receptor ErbB3 in normal human ovary and uterus tissue samples by immunohistochemistry (IHC). ErbB3 is one of the bona fide receptors for NRG1. NRG1 and ErbB3 were colocalized with steroidogenic acute regulatory protein (StAR) in normal human ovarian tissue samples, whereas NRG1 and ErBB3 were colocalized with CD105 (Endoglin) and epithelial cadherin (E-cadherin) in human uterine tissue samples. Our results showed strong immunoreactive signals of NRG1, ErbB3, and StAR in antral follicles, whereas weak or lack of immunoreactive signals in preantral follicles. The strong immunoreactive signals of NRG1 and ErbB3 were observed in granulosa cells and theca interstitial cells of antral follicles. In the uterus, a differential pattern of NRG1, ErbB3, CD105, and E-cadherin expressions were noticed. Interestingly, strong immunoreactive signals of NRG1, ErbB3, CD105, and E-cadherin in the luminal epithelium, and superficial and deep glands of endometrium were observed, whereas weak immunostaining in myometrium was noticed. These findings suggest that NRG1 and ErbB3 are important intracellular autocrine-paracrine anti-inflammatory factors that may govern antral follicular development and uterine implantation. Nothing to Disclose: SB; AR; IC. Sources of Research Support: This study was supported in part by National Institutes of Health Grants 1SC1 GM130544, G12-RR03034, and P50-HD28934. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Grant #C06 RR18386 from NIH/NCRR. Presentation: Friday, June 16, 2023

THU569 Steroid Receptor Coactivator 3 Is Required To Prevent Early Pregnancy Loss In The Mouse

Abstract Disclosure: V. Maurya: None. L. Hai: None. M.M. Szwarc: None. W. San-Pin: None. F.J. DeMayo: None. J.P. Lydon: None. Members of the Steroid Receptor Coactivator (SRC) family enact distinct transcriptional coregulator functions within a broad spectrum of physiologies and pathologies. We previously demonstrated that endometrial SRC-3 is critical for in vitro human endometrial stromal cell decidualization, a critical cellular process in the establishment of the maternofetal interface. To address SRC-3’s in vivo role in endometrial periimplantation biology, we recently generated a SRC-3 conditional knockout mouse model (SRC-3d/d) in which SRC-3 is selectively ablated in cells that express the progesterone receptor. A six-month breeding trial with wild type proven stud males demonstrated that the SRC-3d/d female fails to produce litters (n>10 per genotype). Unlike the SRC-2d/d female, however, both embryo attachment and implantation into the stromal compartment of the SRC-3d/d endometrium are not impaired at gestation day (GD) 5 or 6. While endometrial stromal decidualization initiates with embryo invasion, pregnant SRC-3d/d mice exhibit early pregnancy loss (EPL) by GD-7.5 (before placentation). At GD-6, the SRC-3d/d decidua is significantly smaller than those of sibling controls. Histological analyses reveal extensive decidual cell death in the SRC-3d/d decidua by GD-7, which results in the sudden demise of the embryo. These histological findings are supported by recent genome-wide RNA profiling studies that show a pro-survival role for SRC-3 in this tissue. Based on these results, we conclude that endometrial SRC-3 is critical for maintaining the functionality and survival of the decidual stromal cell and embryo developmental progression to placentation. Therefore, endometrial SRC-3 (and its downstream signaling networks) are predicted to represent new molecular determinants of non-aneuploid EPL, a poorly understood pregnancy complication for which diagnostic and treatment options are limited. Presentation Date: Thursday, June 15, 2023

The IGF1 Signaling Pathway: From Basic Concepts to Therapeutic Opportunities.

Insulin-like growth factor 1 (IGF1) is a peptide growth factor with important functions in multiple aspects of growth, development and metabolism. The biological actions of IGF1 are mediated by the IGF1 receptor (IGF1R), a cell-surface protein that is evolutionarily related to the insulin receptor (InsR). The effects of IGF1 are moderated by a group of binding proteins (IGFBPs) that bind and transport the ligand in the circulation and extracellular fluids. In mechanistic terms, IGF1R function is linked to the MAPK and PI3K signaling pathways. Furthermore, IGF1R has been shown to migrate to cell nucleus, where it functions as a transcriptional activator. The co-localization of IGF1R and MAPK in the nucleus is of major interest as it suggests novel mechanistic paradigms for the IGF1R-MAPK network. Given its potent anti-apoptotic and pro-survival roles, and in view of its almost universal pattern of expression in most types of cancer, IGF1R has emerged as a promising molecular target in oncology. The present review article provides a concise overview of key scientific developments in the research area of IGF and highlights a number of more recent findings, including its nuclear migration and its interaction with oncogenes and tumor suppressors.

P-210 Novel pro-survival role for tryptophan 2,3 dioxygenase 2 in multiple myeloma

P-210 Novel pro-survival role for tryptophan 2,3 dioxygenase 2 in multiple myeloma