Radiotherapy is a mainstay in the management of locally advanced lung cancer; however, intrinsic and acquired radioresistance contribute to poor prognosis. S6K1, a serine/threonine kinase, regulates cell growth, protein synthesis, and survival, and is increased in tumors, which is linked to enhanced survival under therapeutic stress, including radiation. The mechanisms, however, are not fully understood. This study investigates the role of S6K1 in lung cancer radioresistance and the mechanisms involved. Intrinsic radioresistance in lung cancer cells was associated with increased S6K1 activation. Pharmacologic inhibition or genetic deletion of S6K1 enhanced radiosensitivity both in vitro and in vivo, highlighting the therapeutic potential of targeting S6K1. Transcriptomic analysis revealed that S6K1 deletion significantly downregulated STAT3 expression, a transcription factor that promotes radioresistance. S6K1 deletion reduced STAT3 phosphorylation and transcriptional activity, thereby sensitizing lung cancer to radiation. Additionally, radiation exposure or overexpression of a constitutively active S6K1 isoform restored STAT3 activation in S6K1 knockout cells, underscoring the regulatory role of S6K1 in STAT3 signaling. Together, these findings establish a novel S6K1-STAT3 axis that drives radioresistance in lung cancer and suggest that targeting this pathway may enhance radiotherapy efficacy.

IntroductionHepatocellular carcinoma (HCC) is the most prevalent liver cancer and a leading cause of cancer-related deaths worldwide. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) plays a critical role in RNA metabolism, including alternative splicing, which is linked to cancer progression. Our study investigated the role of hnRNPA1 in HCC and its potential as a therapeutic target.MethodsWe analyzed hnRNPA1 expression in HCC tissues compared to non-tumor tissues using RNA-seq and immunohistochemistry. hnRNPA1 was knocked down in Hep G2 cells to assess its impact on cell proliferation, migration, and apoptosis using scratch assays, flow cytometry, qPCR, and Western blot. We also explored the interaction between hnRNPA1 and ZNF207, as well as its splicing effects and downstream signaling pathways by RIP assay, bioinformatics, qPCR and Western blot.ResultshnRNPA1 was significantly upregulated in HCC tissues compared to normal tissues, correlating with poor patient survival. hnRNPA1 knockdown reduced Hep G2 cell proliferation and migration while increasing apoptosis. We identified that hnRNPA1 bound to ZNF207 and regulated its exon 9 skipping, influencing ZNF207 splicing and the PI3K/Akt/mTOR pathway, key regulators of cell growth and survival.ConclusionOur findings demonstrate that hnRNPA1 promotes HCC progression by regulating ZNF207 splicing and the PI3K/Akt/mTOR pathway. hnRNPA1-ZNF207 interaction represents a potential therapeutic target for HCC, providing insights into the molecular mechanisms underlying HCC progression.

Cancer cells uptake excessive nutrients by expressing higher levels of glucose and/or amino acid transporters to meet their increased energy demands. L-type amino acid transporter 1 (LAT1), is regarded as a cancer-specific transporter for the uptake of large neutral amino acids such as L-tryptophan. However, the mechanism by which LAT1 rewires cellular metabolism to promote cancer progression and chemoresistance have not yet been investigated. The protein levels of LAT1, p-PKM2, and p-LDHA were determined in breast cancer tissue arrays by immunohistochemistry staining followed by survival analysis. The orthotopic breast cancer models in mice, syngeneic breast cancer models, and patient-derived xenograft (PDX) mouse models were used to study the effects of LAT1 inhibition in tumor growth and chemoresistance. Steady-state polar metabolite analysis was performed to profile changes in cellular metabolism by LC-MS. The pyruvate and lactate assays as well as the seahorse assay using LAT1 knockdown cells and control cells were conducted to evaluate cellular glycolytic activities. The LAT1 protein levels were positively correlated with poor survival in triple-negative breast cancer (TNBC) patients. LAT1 silencing resulted in reduced TNBC cell viability, proliferation, migration, invasion in vitro, as well as tumor growth in vivo. The knockdown of LAT1 reduced glycolytic activities via activating PKM2 and LDHA, two key glycolytic enzymes essential for cancer cell growth. Mechanistically, we demonstrated that LAT1 promoted de novo NAD + synthesis by facilitating L-tryptophan uptake and upregulating quinolinate phosphoribosyltransferase (QPRT), the rate-limiting enzyme in this pathway. This resulted in an increased cytosolic NAD+/NADH ratio, which enhanced the phosphorylation of pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA), thus promoting TNBC tumor progression. Notably, upregulation of this pathway was observed in primary cells from doxorubicin (Dox)-resistant TNBC patient-derived xenograft (PDX) tumors and in Dox-resistant MDA-MB-231 cells. LAT1 inhibition sensitized resistant cells to Dox-induced cytotoxicity while supplementation of L-Trp/NAD + partially reversed the enhanced sensitivity to Doxorubicin induced by LAT1 knockdown. Furthermore, treatment with a LAT1-specific inhibitor JPH203 synergistically enhanced the efficacy of doxorubicin in TNBC cells. These findings identify a novel role of LAT1 in promoting TNBC progression and chemo-resistance by amplifying the Warburg effect, positioning LAT1 as a promising therapeutic target for TNBC treatment.

BackgroundMelanomas are caused by the malignant transformation of melanocytes. Numerous studies have demonstrated that the tyrosol components of salidroside inhibit tyrosinase activity. The PI3K/Akt/mTOR signaling pathway plays a crucial role in biological pigment synthesis. However, how salidroside achieves its anti-melanoma effect in melanoma by regulating PI3K/Akt/mTOR remains poorly understood. This study aimed to explore the effect of salidroside on PI3K/Akt/mTOR in melanoma, which plays a role in regulating melanogenesis.MethodsNetwork pharmacology was predicted that salidroside may exert an anti-melanoma effect through modulating melanin synthesis functions and signaling pathways. Zebrafish whole-embryo in situ hybridization, RT-qPCR, melanin synthesis and tumorigenesis assays, and were performed to investigate the therapeutic efficacy of salidroside in melanin synthesis. The mechanism of salidroside in anti-melanoma activity was examined by RT-qPCR, Western blot, immunofluorescence, in vivo imaging, immunohistochemistry.ResultsWe confirmed salidroside may exert an anti-melanoma effect through modulating melanin synthesis-related gene expression and PI3K/Akt pathway by Network pharmacology. Furthermore, salidroside slowed melanin synthesis in zebrafish embryos and H2O2-induced B16F10 cells by inhibited oxidative stress. Moreover, we determined the effect of salidroside on anti-melanin synthesis via PI3K/Akt/mTOR pathway in vitro, and western blot results showed that salidroside increased the expression of Nrf2 in the nucleus, as well as inhibited the phosphorylation of mTOR and PI3K/Akt pathway. Finally, intratumoral administration showed salidroside suppressed melanoma growth.ConclusionSalidroside inhibits melanin synthesis and melanoma development most likely by its antioxidant properties and downregulating the PI3K/Akt/mTOR pathway. Our results may provide a novel therapeutic strategy for the treatment of melanoma.

Abstract SLC7A5/LAT1 is a cross-membrane transporter that functions in uptake of large neutral amino acids into cells. Many pathological studies have shown that LAT1 is highly expressed in cancer tissue from various origins such as lung, biliary tract, breast, prostate, bone, and their metastatic legions. High LAT1 expression has been recognized as a significant prognostic marker in various cancers. Our TCGA data analysis showed that the expression level of LAT1 is higher in TNBC tumors compared with normal breast tissues. The higher expression level of LAT1 is linked with poor overall and progression-free survival. Dysregulation of amino acid transporters lead to metabolic reprogramming, which changes intracellular amino acid levels, contributing to the pathogenesis of cancer. However, it remains elusive whether and how LAT1 re-wires the cellular metabolic programs to promote TNBC tumor proliferation. In this study, we showed that inhibition of SLC7A5/LAT1 significantly reduced TNBC cell proliferation, viability, migration capability while promoting apoptosis. Mechanistically, we found significant positive correlations of LAT1 with PKM2 and LDHA in breast cancer tissues, which are key enzymes in mediating glycolysis and lactate fermentation or the Warburg Effect. Knock down LAT1 via siRNA decreased the expression levels of p-PKM2 and p-LDHA in TNBC cells. Tryptophan, a major substrate amino acid transported into cytosol by LAT1, is the precursor for the de novo synthesis of nicotinamide adenine dinucleotide (NAD+). The enzyme quinolinic acid phosphoribosyltransferase (QPRT) catalyzes the conversion from quinolinate, a product of tryptophan degradation, to NAD+. We showed LAT1 inhibition reduced the expression level of QPRT, cytosolic NAD+/NADH ratio as well as lactate production. Addition of L-Tryptophan increased the levels of p-PKM2 and p-LDHA while knockdown of LAT1 reversed tryptophan-mediated increase of PKM2 and LDHA activities, suggesting that LAT1 may regulate PKM2/LDHA through Trp/QPRT/NAD+ pathway for promoting the Warburg Effect. Additionally, we found upregulations of SLC7A5/LAT1, p-PKM2 and p-LDHA in doxorubicin-resistant cells and PDX mouse model compared with their corresponding controls. Collectively, these results provide evidence that overexpression of LAT1 in TNBC drives cancer progression and drug resistance by reinforcing the Warburg Effect, suggesting LAT1 as a promising therapeutic target for addressing TNBC. Our ongoing work will evaluate whether inhibiting LAT1 genetically or pharmacologically reduces xenograft tumor growth and enhances the sensitivities of TNBC resistant cells to chemotherapy in vivo. Our investigation will elucidate the underlying mechanisms involved in these processes. Citation Format: Margot Y. Fedoroff, Lei Zhao, Leili Saeednejad Zanjani, Jun He. SLC7A5/LAT1 promotes the Warburg Effect for TNBC progression through Trp/QPRT/NAD+ pathway [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 3067.

Abstract Given the fast-growing cancer cells, there is an increased demand for large amounts of energy and nucleotides for DNA and RNA synthesis. Thus, targeting nucleotide metabolism, specifically de novo nucleotide biosynthesis, is a promising strategy for cancer treatment. Hyperactivation of Carbamoyl-phosphate synthetase, aspartate transcarbamylase (CAD), a key enzyme catalyzing the first three steps of de novo pyrimidine synthesis, is a common event in tumors and has been associated with chemoresistance in tumors. Our analysis, using TCGA data, revealed that increased expression of CAD is associated with poor prognosis in non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have demonstrated significant benefits to patients with NSCLC harboring EGFR-activating mutations; however, acquired resistance limits their long-term efficacy. Therefore, it remains urgent to discover the underlying mechanisms for discovering novel therapeutic strategies for overcoming TKI resistance. Our previous findings demonstrated that blockade of S6K1 overcomes acquired resistance to EGFR-TKIs in NSCLC. It was reported that S6K1 is involved in de novo pyrimidine biosynthesis. In this study, we aimed to investigate whether CAD mediates S6K1-induced EGFR-TKI resistance in NSCLC. Immunohistochemistry analysis on a tissue microarray of lung adenocarcinoma tissues indicated that increased co-expression of p-CAD and S6K1 proteins is associated with clinicopathological parameters and poor survival outcomes. Our in vitro results showed an increased expression level of CAD and p-CAD proteins in TKI-resistant cells compared with parental cells upon TKI treatment. Knockdown of CAD by siRNA led to sensitizing TKI-resistant cells to osimertinib, as indicated by BrdU cell proliferation assay. Inhibition of S6K1 by a siRNA or specific inhibitors PF-470867 and LY2584702 effectively reduced the phosphorylation levels of CAD protein in TKI-resistant cells, suggesting that S6K1 may serve as an upstream regulator of CAD for TKI resistance. The String database predicted a protein-protein interaction between S6K1 and CAD. Indeed, our co-immunoprecipitation (co-IP) results revealed a direct association between S6K1 and CAD proteins. Importantly, TKI treatment enhanced the binding between S6K1 and CAD. Taken together, these findings provide evidence that CAD may mediate S6K1-mediated EGFR-TKI resistance in NSCLC. Our ongoing and future work will assess whether inhibition of CAD sensitizes resistant cells to TKI in vivo and elucidate the associated mechanism. Citation Format: Leili Saeednejad Zanjani, Lei Zhao, Shaomin Wang, Margot Federoff, Xin Sun, Jun He. Inhibition of CAD, an enzyme of de novo pyrimidine synthesis, to overcome EGFR-TKI resistance in NSCLC [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 7206.

The development of acquired resistance to small molecule tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) signaling has hindered their efficacy in treating non-small cell lung cancer (NSCLC) patients. Our previous study showed that constitutive activation of the 70 kDa ribosomal protein S6 kinase 1 (S6K1) contributes to the acquired resistance to EGFR-TKIs in NSCLC cell lines and xenograft tumors in nude mice. However, the regulatory mechanisms underlying S6K1 constitutive activation in TKI-resistant cancer cells have not yet been explored. In this study, we recapitulated this finding by taking advantage of a gefitinib-resistant patient-derived xenograft (PDX) model established through a number of passages in mice treated with increasing doses of gefitinib. The dissociated primary cells from the resistant PDX tumors (PDX-R) displayed higher levels of phosphor-S6K1 expression and were resistant to gefitinib compared to cells from passage-matched parental PDX tumors (PDX-P). Both genetic and pharmacological inhibition of S6K1 increased sensitivity to gefitinib in PDX-R cells. In addition, both total and phosphorylated mechanistic target of rapamycin kinase (MTOR) levels were upregulated in PDX-R and gefitinib-resistant PC9G cells. Knockdown of MTOR by siRNA decreased the expression levels of total and phosphor-S6K1 and increased sensitivity to gefitinib in PDX-R and PC9G cells. Moreover, a transcription factor ELK1, which has multiple predicted binding sites on the MTOR promoter, was also upregulated in PDX-R and PC9G cells, while the knockdown of ELK1 led to decreased expression of MTOR and S6K1. The chromatin immunoprecipitation (ChIP)-PCR assay showed the direct binding between ELK1 and the MTOR promoter, and the luciferase reporter assay further indicated that ELK1 could upregulate MTOR expression through tuning up its transcription. Silencing ELK1 via siRNA transfection improved the efficacy of gefitinib in PDX-R and PC9G cells. These results support the notion that activation of ELK1/MTOR/S6K1 signaling contributes to acquired resistance to gefitinib in NSCLC. The findings in this study shed new light on the mechanism for acquired EGFR-TKI resistance and provide potential novel strategies by targeting the ELK1/MTOR/S6K1 pathway.

e21029 Background: RET fusions occur in 2-3% of lung cancer cases, and there are currently two FDA-approved RET-specific inhibitors (RETi), selpercatinib and pralsetinib. RET fusions are heterogeneous and the impact of specific fusion partners and their unique breakpoints on drug sensitivity is poorly understood. Moreover, while patients may be initially sensitive to RETi treatment, therapeutic resistance eventually emerges, and studies have shown that RET-dependent mechanisms of resistance include acquired resistance mutations that alter drug binding. We sought to investigate mechanisms of sensitivity and resistance to RET inhibitors in RET-fusion positive lung cancer in effort to improve treatment options and clinical outcomes in this patient population. Methods: We characterized the landscape of RET-fusions and -mutations in non-small cell lung cancer (NSCLC). KIF5B-RET, CCDC6-RET, and NCOA4-RET were the most frequently observed RET fusions in NSCLC. To investigate the drug sensitivity profile of individual RET fusion variants, we generated a panel of Ba/F3 cells expressing various RET fusion variants and evaluated in vitro sensitivity to RETi including selpercatinib, pralsetinib, vandetinib, cabozantinib, TPX-0046, lovatinib, and ponatinib. We observed that unique RET fusions impart differential sensitivities to RET inhibitors. For example, cells expressing KIF-RET (K15, R12) were highly sensitive to selpercatinib, pralsetinib, and ponatinib, whereas cells expressing KIF-RET (K22, R12) were likewise sensitive to selpercatinib and pralsetinib but were less sensitive to ponatinib. We next established a cell line (MDA-L-30) from a patient harboring a RET-fusion (KIF-RET; K15, R12) and observed a RETi sensitivity profile similar to that observed using the BaF3 system. Results: Next, we employed the LentiMutate approach to rapidly identify genomic mechanisms of RETi resistance. In cells expressing the KIF5B-RET (K15, R12) fusion, the G810S mutation was associated with acquired resistance to both selpercatinib and pralsetinib, consistent with prior reports and clinical observations. However, V804M/E and M918T mutations were associated with acquired resistance to selpercatinib but not pralsetinib, and L730I/V and E732K mutations were associated with resistance to pralsetinib but not selpercatinib. Conclusions: Taken together, these data indicate that individual RET fusion variants with diverse fusion partner breakpoints have distinct drug sensitivity profiles and that secondary resistance mutations may be non-overlapping between RETi. These findings support the need to comprehensively characterize RET-dependent mechanisms of resistance in order to provide therapeutic recommendations for treating RET-fusion driven cancers.

Metabolic reprogramming wherein the cancer cells exhibit altered energetics is a hallmark of cancer. Although recent discoveries have enhanced our understanding of tumor metabolism, the therapeutic utility of targeting tumor metabolism is not yet realized. Glutamine, a non-essential amino acid, plays a critical role in regulating tumor metabolism and provides an alternative tumor energy source. In this study, we investigate the molecular mechanism regulated by glutamine and elucidate if targeting glutamine metabolism would enhance the efficacy of cancer chemotherapy. Using clonogenic and cell cycle analysis, we found that deprivation of glutamine suppress the growth of cancer cells. Mechanistically we demonstrate that glutamine stabilizes myc by preventing its ubiquitination through alpha-ketoglutarate. Inhibition of glutamine metabolism enhanced the sensitivity of tumor cells to chemotherapeutic agent paclitaxel. Our results delineate the mechanism behind glutamine-induced myc stabilization, and they provide a viable strategy to target cancer with a glutamine metabolism inhibitor in the clinic.

Cancer cells have an imbalance in oxidation-reduction (redox) homeostasis. Understanding the precise mechanisms and the impact of the altered redox microenvironment on the immunologic reaction to tumors is limited. We isolated exosomes from ovarian cancer cells through ultracentrifuge and characterized by Western-blots and Nanoparticle Tracking Analysis. 2D, 3D-coculture tumor model, and 3D live cell imaging were used to study the interactions between tumor cells, macrophages and CD3 T cells in vitro. The role of exosomal miR-155-5p in tumor growth was evaluated in xenograft nude mice models and immune-competent mice models. Flow cytometry and flow sorting were used to determine the expression levels of miR-155-5p and PD-L1 in ascites and splenic macrophages, and the percentages of CD3 T cells subpopulations. The elevation of reactive oxygen species (ROS) greatly downregulated exosomal miR-155-5p expression in tumor cells. Neutralization of ROS with N-acetyl-L-cysteine (NAC) increased the levels of miR-155-5p in tumor exosomes that were taken up by macrophages, leading to reduction of macrophage migration and tumor spheroid infiltration. We further found that programmed death ligand 1 (PD-L1) is a functional target of miR-155-5p. Co-culture of macrophages pre-treated with NAC-derived tumor exosomes or exosomal miR-155-5p with T-lymphocytes leading to an increased percentage of CD8+ T-lymphocyte and a decreased CD3+ T cell apoptosis through PD-L1 downregulation. Tumor growth in nude mice was delayed by treatment with NAC-derived tumor exosomes. Delivery of tumor exo-miR-155-5p in immune-intact mice suppressed ovarian cancer progression and macrophage infiltration, and activated CD8+ T cell function. It is of note that exo-miR-155-5p inhibited tumor growth more potently than the PD-L1 antibody, suggesting that in addition to PD-L1, other pathways may also be targeted by this approach. Our findings demonstrate a novel mechanism, ROS-induced down-regulation of miR-155-5p, by which tumors modulate the microenvironment that favors tumor growth. Understanding of the negative impact of ROS on the tumor immune response will improve current therapeutic strategies. Targeting miR-155-5p can be an alternative approach to prevent formation of an immunosuppressive TME through downregulation of PD-L1 and other immunosuppressive factors.

Chronic exposure to hexavalent chromium compounds [Cr(VI)] is associated with an increased risk of cancers, but the molecular mechanisms remain to be elucidated. In this study, we found that CXCL5 levels in peripheral blood monocytes (PBMCs) and plasma from workers with occupational exposure to Cr(VI) were dramatically upregulated compared to non-exposure healthy subjects, and plasma C-X-C Motif Chemokine Ligand 5 (CXCL5) CXCL5 levels were positively correlated with Cr concentrations in subjects' toenails. Zinc chromate exposed mice showed higher levels of CXCL5 and its receptor CXCR2 in lung tissues, and in PBMCs. Similar CXCL5 upregulation was evident in Cr(VI)-induced transformed (Cr-T) cells with long-term Cr(VI) treatment. Mechanistic studies showed that elevated CXCL5 expression levels were regulated by Cr(VI)-induced histone modifications and DNA hypomethylation, and that the c-Myc/p300 complex was a key upstream regulator of histone H3 acetylation. CXCL5 overexpression promoted Cr(VI)-induced the epithelial to mesenchyme transition (EMT) by upregulating zinc finger E-box binding homeobox 1 (ZEB1) to promote tumor development. Our findings identify a novel mechanism by which CXCL5 is upregulated and promotes EMT and carcinogenesis upon chronic Cr(VI) exposure. Our work also implies that CXCL5 mRNA and protein levels will elevate in PBMCs and serum after occupational Cr(VI) exposure, which may be a potential target and biomarker for cancer prevention and health surveillance among populations exposed to Cr(VI).

The metabolic abnormality observed in tumors is characterized by the dependence of cancer cells on glycolysis for their energy requirements. Cancer cells also exhibit a high level of reactive oxygen species (ROS), largely due to the alteration of cellular bioenergetics. A highly coordinated interplay between tumor energetics and ROS generates a powerful phenotype that provides the tumor cells with proliferative, antiapoptotic, and overall aggressive characteristics. In this review article, we summarize the literature on how ROS impacts energy metabolism by regulating key metabolic enzymes and how metabolic pathways e.g., glycolysis, PPP, and the TCA cycle reciprocally affect the generation and maintenance of ROS homeostasis. Lastly, we discuss how metabolic adaptation in cancer influences the tumor's response to chemotherapeutic drugs. Though attempts of targeting tumor energetics have shown promising preclinical outcomes, the clinical benefits are yet to be fully achieved. A better understanding of the interaction between metabolic abnormalities and involvement of ROS under the chemo-induced stress will help develop new strategies and personalized approaches to improve the therapeutic efficiency in cancer patients.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant forms of cancer. Lack of effective treatment options and drug resistance contributes to the low survival among PDAC patients. In this study, we investigated the metabolic alterations in pancreatic cancer cells that do not respond to the EGFR inhibitor erlotinib. We selected erlotinib-resistant pancreatic cancer cells from MiaPaCa2 and AsPC1 cell lines. Metabolic profiling of erlotinib-resistant cells revealed a significant downregulation of glycolytic activity and reduced level of glycolytic metabolites compared to the sensitive cells. The resistant cells displayed elevated expression of the pentose phosphate pathway (PPP) enzymes involved in ROS regulation and nucleotide biosynthesis. The enhanced PPP elevated cellular NADPH/NADP+ ratio and protected the cells from reactive oxygen species (ROS)-induced damage. Inhibition of PPP using 6-aminonicotinamide (6AN) elevated ROS levels, induced G1 cell cycle arrest, and sensitized resistant cells to erlotinib. Genetic studies identified elevated PPP enzyme glucose-6-phosphate dehydrogenase (G6PD) as an important contributor to erlotinib resistance. Mechanistically, our data showed that upregulation of inhibitor of differentiation (ID1) regulates G6PD expression in resistant cells thus contributing to altered metabolic phenotype and reduced response to erlotinib. Together, our results highlight an underlying role of tumor metabolism in PDAC drug response and identify G6PD as a target to overcome drug resistance.

Abstract Background and Objective: Genetic and metabolic alterations in cancer cells promote the sustained oxidative stress as reflected by excessive production of reactive oxygen species (ROS). We and other groups have identified that the ROS-sensitive microRNAs regulate the cancer cell response to ROS. However, it has yet to be determined how these miRNAs convey the ROS signal to downstream effectors and thus produce biological consequences. Accumulative evidence indicates that tumor-derived exosomes are emerging mediators of tumorigenesis. In this study, we aimed to investigate how ROS convey the oncogenic signals through cellular interactions via exosomes within the tumor microenvironment. Methods and Results: Through NanoString nCounter miRNA analysis, we identified miR-155-5p as the most abundant miRNAs enriched in the exosomes derived from epithelial ovarian cancer (EOC) cells treated with a ROS scavenger N-acetylcysteine (NAC). Mechanistically, tumor exosomal miR-155-5p was transferred to tumor-associated macrophages (TAM), directly targeting PD-L1 to inhibit the infiltration of TAM into tumor microenvironment. The role of tumor-derived exosomal miR-155-5p was determined by co-culture experiments and gain or loss of function studies in vitro, and confirmed in xenograft mouse models in nude mice and immunocompetent mice. In addition, we established a 3D-3-culture system to entail the co-culture of NAC-treated EOC cell spheroids, TAM and T cells. We found that CD8+ T-cell ratio was increased and production of IFN-γ, IL-2, and TNF-α was promoted. What's more, the immunocompetent EOC mouse model was established and treated with NAC-modified mouse ovarian cancer cell-released exosomes. The results showed that NAC-modified tumor growth was significantly reduced compared to control group. Further analysis showed that miR-155-5p increased in TAM, while PD-L1 expression levels decreased in macrophages from tumors and spleens. Moreover, the T cell function was also augmented as evaluated by the expression of Ki-67 and Granzyme B on CD8+ T cells. Conclusion: In conclusion, our study reveals a novel mechanism underlying PD-L1 expression in EOC. Ovarian cancer cells with excessive ROS levels release exosomes that mediate the interaction between cancer cells and immune cells; it is this interplay that produces an immunosuppressive tumor microenvironment by upregulation of PD-L1 in macrophages, and eventually contributes to the progress of EOC through the ROS/miR-155-5p/PD-L1 pathway. Citation Format: Xiang Li, Gao-chan Wang, Jun He. Excessive ROS in ovarian cancer cells impairs anti-tumor capacities of immune cells through exosomal miR-155/PD-L1 pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1700.

The development of resistance to EGFR Tyrosine kinase inhibitors (TKIs) in NSCLC with activating EGFR mutations is a critical limitation of this therapy. In addition to genetic alterations such as EGFR secondary mutation causing EGFR-TKI resistance, compensatory activation of signaling pathways without interruption of genome integrity remains to be defined. In this study, we identified S6K1/MDM2 signaling axis as a novel bypass mechanism for the development of EGFR-TKI resistance. The observation of S6K1 as a candidate mechanism for resistance to EGFR TKI therapy was investigated by interrogation of public databases and a clinical cohort to establish S6K1 expression as a prognostic/predictive biomarker. The role of S6K1 in TKI resistance was determined in in vitro gain-and-loss of function studies and confirmed in subcutaneous and orthotopic mouse lung cancer models. Blockade of S6K1 by a specific inhibitor PF-4708671 synergistically enhanced the efficacy of TKI without showing toxicity. The mechanistic study showed the inhibition of EGFR caused nuclear translocation of S6K1 for binding with MDM2 in resistant cells. MDM2 is a downstream effector of S6K1-mediated TKI resistance. Taken together, we present evidence for the reversal of resistance to EGFR TKI by the addition of small molecule S6K1/MDM2 antagonists that could have clinical benefit.

Hexavalent chromium [Cr(VI)] is a known occupational and environmental contaminant and carcinogen, but new mechanisms of Cr(VI)-induced carcinogenesis remain to be elucidated. In this study, we found that expression of miR-143 is decreased, whereas that of Interleukin 6 (IL-6) is increased in blood samples of Cr(VI)-exposing workers compared with corresponding unexposed workers. In addition, IL-6 was increased in human bronchial epithelial cells (BEAS-Cr) exposed to Cr(VI) compared with unexposed BEAS-2B cells. To further investigate the mechanisms by which Cr(VI) promotes these changes, we assessed the effects of miR-143 on gene expression and found that miR-143 suppressed expression of IL-6, HIF-1α and NF-κB p65, and that inhibiting miR-143 promoted expression of IL-6, HIF-1α and NF-κB p65. Interestingly, IL-6 regulated expression of HIF-1α, and HIF-1α transcriptionally regulated expression of IL-6. Experiments in animals showed that miR-143 inhibited tumor growth and angiogenesis by regulating IL-6/HIF-1α and downstream signaling pathways in vivo. These outcomes support the hypothesis that the miR-143/IL-6/HIF-1α pathway functions to regulate Cr(VI)-induced carcinogenesis.

Inorganic arsenic is an environmental carcinogen that poses a major global public health risk. A high percentage of drinking water from wells in the U.S. contains higher-than-normal levels of arsenic, suggesting an increased risk of arsenic-induced deleterious effects. In addition to primary preventive measures, therapeutic strategies need to effectively address and integrate multiple molecular mechanisms underlying arsenic-induced carcinogenesis. We previously showed that the loss of miR-199a-5p in arsenic-transformed cells is pivotal to promote arsenic-induced angiogenesis and tumor growth in lung epithelial cells. In this study, we further showed that subacute or chronic exposure to arsenic diminished miR-199a-5p levels largely due to DNA methylation, which was achieved by increased DNA methyltransferase-1 (DNMT1) activity, mediated by the formation of specific protein 1 (Sp1)/DNMT1 complex. In addition to the DNA hypermethylation, arsenic exposure also repressed miR-199a transcription through a transcriptional repressor Sp1. We further identified an association between miR-199a-5p repression and the arsenic-mediated energy metabolic shift, as reflected by mitochondria defects and a switch to glycolysis, in which a glycolytic enzyme pyruvate kinase 2 (PKM2) was a functional target of miR-199a-5p. Taken together, the repression of miR-199a-5p through both Sp1-dependent DNA methylation and Sp1 transcriptional repression promotes an arsenic-mediated metabolic shift from mitochondria respiration to aerobic glycolysis via PKM2.

Glioblastoma multiforme (GBM) is one of the most hypoxic tumors of the central nervous system. Although temozolomide (TMZ) is an effective clinical agent in the GBM therapy, the hypoxic microenvironment remains a major barrier in glioma chemotherapy resistance, and the underlying mechanisms are poorly understood. Here, we find hypoxia can induce the protective response to mitochondrion via HIF-1α-mediated miR-26a upregulation which is associated with TMZ resistance in vitro and in vivo. Further, we demonstrated that HIF-1α/miR-26a axis strengthened the acquisition of TMZ resistance through prevention of Bax and Bad in mitochondria dysfunction in GBM. In addition, miR-26a expression levels negatively correlate with Bax, Bad levels, and GBM progression; but highly correlate with HIF-1α levels in clinical cancer tissues. These findings provide a new link in the mechanistic understanding of TMZ resistance under glioma hypoxia microenvironment, and consequently HIF-1α/miR-26a/Bax/Bad signaling pathway as a promising adjuvant therapy for GBM with TMZ.

Hirschsprung’s disease (HSCR) is a rare congenital disease caused by impaired proliferation and migration of neural crest cells. We investigated changes in expression of microRNAs (miRNAs) and the genes they regulate in tissues of patients with HSCR. Quantitative real-time PCR and immunoblot analyses were used to measure levels of miRNA, mRNAs, and proteins in colon tissues from 69 patients with HSCR and 49 individuals without HSCR (controls). Direct interactions between miRNAs and specific mRNAs were indentified in vitro, while the function role of miR218-1 was investigated by using miR-218 transgenic mice. An increased level of miR-218-1 correlated with increased levels of SLIT2 and decreased levels of RET and PLAG1 mRNA and protein. The reductions in RET and PLAG1 by miR-218-1 reduced proliferation and migration of SH-SY5Y cells. Overexpression of the secreted form of SLIT2 inhibited cell migration via binding to its receptor ROBO1. Bowel tissues from miR-218-1 transgenic mice had nerve fibre hyperplasia and reduced numbers of gangliocytes, compared with wild-type mice. Altered miR-2181 regulation of SLIT2, RET and PLAG1 might be involved in the pathogenesis of HSCR.

Epithelial-mesenchymal transition (EMT) has been recognized as a key element of cell migration and invasion in lung cancer; however, the underlying mechanisms are not fully elucidated. Recently, emerging evidence suggest that miRNAs have crucial roles in control of EMT and EMT-associated traits such as migration, invasion and chemoresistance. Here, we found that miR-218 expression levels were significantly downregulated in lung cancer tissues compared with adjacent non-cancerous tissues, and the levels of miR-218 were significantly associated with histological grades and lymph node metastasis. Overexpression of miR-218 inhibited cell migration and invasion as well as the EMT process. Of particular importance, miR-218 was involved in the metastatic process of lung cancer cells in vivo by suppressing local invasion and distant colonization. We identified Slug and ZEB2 as direct functional targets of miR-218. Inverse correlations were observed between miR-218 levels and Slug/ZEB2 levels in cancer tissue samples. In addition, overexpression of miR-218 in H1299 increased chemosensitivity of cells to cisplatin treatment through suppression of Slug and ZEB2. These findings highlight an important role of miR-218 in the regulation of EMT-related traits and metastasis of lung cancer in part by modulation of Slug/ZEB2 signaling, and provide a potential therapeutic strategy by targeting miR-218 in NSCLC.