Head and Neck Squamous Cell Carcinoma (HNSCC) imposes a significant health burden, necessitating innovative therapeutic strategies to enhance treatment efficacy. Current treatments, such as surgery, radiation, and chemotherapy, have limited effectiveness and yield severe side effects, emphasizing the need for targeted therapies. We have focused on DNA polymerase beta (Polβ) and its roles in replication stress, cellular responses to DNA damaging-therapies, and DNA damage response modifiers. Our investigations reveal a regulatory role for base excision repair (BER) proteins, including Polβ, in the cellular response to inhibitors of poly(ADP-ribose) glycohydrolase (PARG), an enzyme involved in poly(ADP-ribose) (PAR) degradation. The inhibition of PARG, in HNSCC cells, elicits replication stress and activates the PAR-induced S-phase/ATR checkpoint, leading to a block to replication, cell cycle arrest, and the onset of apoptosis. However, Polβ overexpression mitigates this response, reducing replication-stress-induced PAR foci formation, suggesting a modulation of replication checkpoint activation. We found that PARG inhibitor treatment is ineffective on HNSCC cells that overexpress Polβ, implying that the PARG inhibitor-induced PAR and apoptotic response is dependent on the level of Polβ. Further, our in vitro experiments demonstrate that combining PARG and ATR/CHK1 inhibitors overcomes Polβ-mediated treatment resistance in HNSCC cells, producing synergistic effects compared to the individual treatment conditions. Our findings suggest a possible treatment paradigm for HNSCC, employing ATR or CHK1 inhibitors in combination with PARG inhibitors. This strategy offers a promising path for more effective HNSCC treatments, potentially overcoming Polβ-related resistance.

BackgroundIn breast cancer, ErbB receptors play a critical role, and overcoming drug resistance remains a major challenge in the clinic. However, intricate regulatory mechanisms of ErbB family genes are poorly understood. Here, we demonstrate SON as an ErbB-regulatory splicing factor and a novel therapeutic target for ErbB-positive breast cancer.MethodsSON and ErbB expression analyses using public database, patient tissue microarray, and cell lines were performed. SON knockdown assessed its impact on cell proliferation, apoptosis, kinase phosphorylation, RNA splicing, and in vivo tumour growth. RNA immunoprecipitation was performed to measure SON binding.ResultsSON is highly expressed in ErbB2-positive breast cancer patient samples, inversely correlating with patient survival. SON knockdown induced intron retention in selective splice sites within ErbB2 and ErbB3 transcripts, impairing effective RNA splicing and reducing protein expression. SON disruption suppressed downstream kinase signalling of ErbB2/3, including the Akt, p38, and JNK pathways, with increased vulnerability in ErbB2-positive breast cancer cells compared to ErbB2-negative cells. SON silencing in ErbB2-positive breast cancer xenografts led to tumour regression in vivo.ConclusionWe identified SON as a novel RNA splicing factor that plays a critical role in regulating ErbB2/3 expression, suggesting SON is an ideal therapeutic target in ErbB2-positive breast cancers.

The mammalian STE 20-like protein kinase 4 (MST4) gene is highly expressed in several cancer types, but little is known about the role of MST4 in breast cancer, and the function of MST4 during epithelial-mesenchymal transition (EMT) has not been fully elucidated. Here we report that overexpression of MST4 in breast cancer results in enhanced cell growth, migration, and invasion, whereas inhibition of MST4 expression significantly attenuates these properties. Further study shows that MST4 promotes EMT by activating Akt and its downstream signaling molecules such as E-cadherin/N-cadherin, Snail, and Slug. MST4 also activates AKT and its downstream pro-survival pathway. Furthermore, by analyzing breast cancer patient tissue microarray and silicon datasets, we found that MST4 expression is much higher in breast tumor tissue compared to normal tissue, and significantly correlates with cancer stage, lymph node metastasis and a poor overall survival rate (p < 0.05). Taken together, our findings demonstrate the oncogenic potential of MST4 in breast cancer, highlighting its role in cancer cell proliferation, migration/invasion, survival, and EMT, suggesting a possibility that MST4 may serve as a novel therapeutic target for breast cancer.

We previously found that QNBC tumors are more frequent in African Americans compared to TNBC tumors. To characterize this subtype further, we sought to determine the miRNA-mRNA profile in QNBC patients based on race. Both miRNA and mRNA expression data were analyzed from TCGA and validated using datasets from the METABRIC, TCGA proteomic, and survival analysis by KMPLOT. miRNA-mRNAs which include FOXA1 and MYC (mir-17/20a targets); GATA3 and CCNG2 (mir-135b targets); CDKN2A, CDK6, and B7-H3 (mir-29c targets); and RUNX3, KLF5, IL1-β, and CTNNB1 (mir-375 targets) were correlated with basal-like and immune subtypes in QNBC patients and associated with a worse survival. Thus, QNBC tumors have an altered gene signature implicated in racial disparity and poor survival.

Metabolism of polyunsaturated fatty acids results in the formation of hydroxylated fatty acids that can be further oxidized by dehydrogenases, often resulting in the formation of electrophilic, α,β-unsaturated ketone containing fatty acids. As electrophiles are associated with redox signaling, we sought to investigate the metabolism of the oxo-fatty acid products in relation to their double bond architecture. Using an untargeted liquid chromatography mass spectrometry approach, we identified mono- and di-saturated products of the arachidonic acid-derived 11-oxoeicosatetraenoic acid (11-oxoETE) and mono-saturated metabolites of 15-oxoETE and docosahexaenoic acid-derived 17-oxodocosahexaenoinc acid (17-oxoDHA) in both human A549 lung carcinoma and umbilical vein endothelial cells. Notably, mono-saturated oxo-fatty acids maintained their electrophilicity as determined by nucleophilic conjugation to glutathione while a second saturation of 11-oxoETE resulted in a loss of electrophilicity. These results would suggest that prostaglandin reductase 1 (PTGR1), known only for its reduction of the α,β-unsaturated double bond, was not responsible for the saturation of oxo-fatty acids at alternative double bonds. Surprisingly, knockdown of PTGR1 expression by shRNA confirmed its participation in the formation of 15-oxoETE and 17-oxoDHA mono-saturated metabolites. Furthermore, overexpression of PTGR1 in A549 cells increased the rate and total amount of oxo-fatty acid saturation. These findings will further facilitate the study of electrophilic fatty acid metabolism and signaling in the context of inflammatory diseases and cancer where they have been shown to have anti-inflammatory and anti-proliferative signaling properties.

Elevated expression of the DNA damage response proteins PARP1 and poly(ADP-ribose) glycohydrolase (PARG) in glioma stem cells (GSCs) suggests that glioma may be a unique target for PARG inhibitors (PARGi). While PARGi-induced cell death is achieved when combined with ionizing radiation, as a single agent PARG inhibitors appear to be mostly cytostatic. Supplementation with the NAD+ precursor dihydronicotinamide riboside (NRH) rapidly increased NAD+ levels in GSCs and glioma cells, inducing PARP1 activation and mild suppression of replication fork progression. Administration of NRH+PARGi triggers hyperaccumulation of poly(ADP-ribose) (PAR), intra S-phase arrest and apoptosis in GSCs but minimal PAR induction or cytotoxicity in normal astrocytes. PAR accumulation is regulated by select PARP1- and PAR-interacting proteins. The involvement of XRCC1 highlights the base excision repair pathway in responding to replication stress while enhanced interaction of PARP1 with PCNA, RPA and ORC2 upon PAR accumulation implicates replication associated PARP1 activation and assembly with pre-replication complex proteins upon initiation of replication arrest, the intra S-phase checkpoint and the onset of apoptosis.

Abstract Purpose: Recently, we have demonstrated that triple-negative breast tumors (TNBC) that lack the expression of androgen receptor are quadruple-negative tumors (QNBC) and have increased aggressiveness. Additionally, we determined that this is due to a unique gene signature in the QNBC subtype. However, whether this gene signature is associated with a unique miRNA expression and differential in African American (AA) women compared to Caucasian (CA) women has not been determined. Therefore, the purpose of this study is to determine the miRNA expression in presence or absence of androgen receptor in both African American and Caucasian QNBC patients. Methods: miRNA level 3 sequencing files were originally downloaded on June 01 2016 from TCGA for 925 breast cancer patients. The miRNA sequence files (RPKM) and gene expression files (FPKM UQ) were obtained for normal and tumor samples for these patients. Median value of AR FPKM UQ was used as cut-off and divided patient group as AR(+ve/-ve). Log2 fold change values were calculated by comparing AR-negative and AR-positive patients. P-value was calculated using two-tail t-test. GSE19783 (mRNA, miRNA data) was also obtained and was used as validating dataset. Results: Out of 925 patients, 101 patients have TNBC. Among TNBC patients, 107 are QNBC and 8 patients are AR +ve TNBC. Reads per million for 1046 miRNAs were considered for this study.40 miRNAs showed differential expression in QNBC. (p-value and lt 0.05). All BC patients were organized based on race (CA/AA), stage (I/II/III, IV) and subtype (Luminal, Her2type, TNBC).We have found that hsa-mir-452 and hsa-mir-937 expression was high in AR-negative, hsa-mir-190b expression was low in AR -ve African American women. miRNAs expression was changed in QNBC and correlated to AR (hsa-mir-577, hsa-mir-135b and hsa-mir-18a). hsa-mir-375 expression was low in QNBC.hsa-mir-150, hsa-mir-181a-2, hsa-mir-500a, hsa-let-7d, hsa-mir-92a-1, hsa-mir-92a-2, hsa-mir-20a, hsa-mir-17, hsa-mir-30a, hsa-mir-210, hsa-mir-455, hsa-mir-130a are expression was changed in African American women with QNBC. Hsa-mir-3607 is low in African American women, irrespective of AR expression. These results are in process of validation using independent dataset. Discussion/Conclusion: Standard treatment of breast cancer relies on reliable assessment by IHC analysis of ER, PR, and HER2. Our results suggest that the heterogeneity of TNBC is at least partially associated with the presence or absence of AR expression, suggesting that QNBC should be considered as a clinically relevant breast cancer subtype. IHC analysis of AR appears to be a practical assay to determine the most aggressive TNBC subtypes and identifies tumors that could benefit from available targeted therapies. miRNA isoform or novel miRNA needs to be discovered to further investigate the gene regulation for QNBC patients. Citation Format: Anusha Angajala, Raymond Hughley, Windy DeanColomb, Shweta Tripathi, Ming Tan, Clayton Yates. Identification of differentially expressed microRNAs in African American women with quadruple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4769.

PURPOSE: Despite the availability of current standards of care treatments for triple negative breast cancer (TNBC), many patients still die from this disease. Quadruple negative tumors, which are TNBC tumors that lack androgen receptor (AR), represent a more aggressive subtype of TNBC; however, the molecular features are not well understood. METHODS: Immunohistochemistry of estrogen receptor (ER), progesterone receptor (PR), HER2, and AR was determined in 244 primary and 630 recurrent/metastatic site biopsies. Expression was correlated with a panel of 25 cancer-related genes and proteins by IHC and in situ hybridization (ISH). RESULTS: We observed that 80.2% (65 of 81) of primary TNBC tumors and 75.7% (159 of 210) of recurrent/metastatic TNBC tumors are QNBC. Bivariate fit analysis demonstrated that QNBC (n = 224) significantly (P < .03) correlated with younger aged patients at initial biopsy compared to AR positive TNBC patients (n = 51). In paired primary tissue samples and primary to recurrent/metastatic samples, at least 70% Luminal, HER2 enriched, and QNBC subtype did not change molecular profile. But, TNBC seems to be the "unstable" subtype. Within the total cohort, discordance in molecular profiles was identified in both synchronous (20%) and asynchronous (21%) intra-individual analyses. Irrespective of sample type, (Synchronous or Asynchronous), QNBC demonstrated higher concordant than TNBC. IHC and ISH results of the cancer related genes, demonstrated that gene/protein expression differ by molecular profile: TNBC (HR-/HER2-, AR+) and QNBC (HR-/HER2-, AR-). IHC in metastatic tumors, showed that the percentage of tumors positive of EGFR were higher, while PTEN and TLE3 were lower in QNBC compared to TNBC. CONCLUSION: Standard treatment of Breast Cancer (BC) relies on reliable assessment by IHC analysis of ER, PR, and HER2. Our analyses suggest that the heterogeneity of TNBC is at least partially associated with the presence or absence of AR expression, suggesting that QNBC should be considered as a clinically relevant BC subtype. IHC analysis of AR appears to be a practical assay to determine the most aggressive TNBC subtypes and identifies tumors that could benefit from available targeted therapies.

Lack of immune system cells or impairment in differentiation of immune cells is the basis for many chronic diseases. Metabolic changes could be the root cause for this immune cell impairment. These changes could be a result of altered transcription, cytokine production from surrounding cells, and changes in metabolic pathways. Immunity and mitochondria are interlinked with each other. An important feature of mitochondria is it can regulate activation, differentiation, and survival of immune cells. In addition, it can also release signals such as mitochondrial DNA (mtDNA) and mitochondrial ROS (mtROS) to regulate transcription of immune cells. From current literature, we found that mitochondria can regulate immunity in different ways. First, alterations in metabolic pathways (TCA cycle, oxidative phosphorylation, and FAO) and mitochondria induced transcriptional changes can lead to entirely different outcomes in immune cells. For example, M1 macrophages exhibit a broken TCA cycle and have a pro-inflammatory role. By contrast, M2 macrophages undergo β-oxidation to produce anti-inflammatory responses. In addition, amino acid metabolism, especially arginine, glutamine, serine, glycine, and tryptophan, is critical for T cell differentiation and macrophage polarization. Second, mitochondria can activate the inflammatory response. For instance, mitochondrial antiviral signaling and NLRP3 can be activated by mitochondria. Third, mitochondrial mass and mobility can be influenced by fission and fusion. Fission and fusion can influence immune functions. Finally, mitochondria are placed near the endoplasmic reticulum (ER) in immune cells. Therefore, mitochondria and ER junction signaling can also influence immune cell metabolism. Mitochondrial machinery such as metabolic pathways, amino acid metabolism, antioxidant systems, mitochondrial dynamics, mtDNA, mitophagy, and mtROS are crucial for immune functions. Here, we have demonstrated how mitochondria coordinate to alter immune responses and how changes in mitochondrial machinery contribute to alterations in immune responses. A better understanding of the molecular components of mitochondria is necessary. This can help in the development of safe and effective immune therapy or prevention of chronic diseases. In this review, we have presented an updated prospective of the mitochondrial machinery that drives various immune responses.

Introduction: Neurogenic hypertension is characterized by increased blood pressure, neuroinflammation, and neuronal oxidative stress. Thus, its pathophysiology dictates a unique therapeutic strategy. It is established that Hibiscus sabdariffa (HS) plays a beneficial role in decreasing blood pressure, yet the role of HS as an anti-oxidant defense system in neurogenic hypertension has not been investigated. Our objective was to generate an in vitro model of neuronal oxidative stress to test the hypothesis that HS creates a neuroprotective antioxidant defense system. Methods: To create a model of oxidative stress, SY5Y, neuroblastoma cells, were treated with 32uM hydrogen peroxide. To determine the effect of methanol-extracted HSE (50 and 100ug n ≥ 8) in this model of oxidative stress, SY5Y cells were treated with vehicle-PBS, HSE (24hrs), pretreated with HSE (24hrs) and then treated with H2O2 (8hrs). Using molecular techniques markers of oxidative stress were measured and percentages were out of 100. Data was normalized to the control and analyzed by one way ANOVA (p value < .05 was considered significant). Results: HSE significantly increases cell viability (50 or 100ug > 99%) compared to H2O2 alone (43%). HSE significantly decreases ROS generation (113%-50ug versus 89%-100ug) compared to H2O2 (120%). HSE significantly decreases lipid peroxidation (11.3%-50ug versus 10.2%-100ug) compared to H2O2 (135%). HSE significantly increases GSH content (72%-50ug versus 91%-100ug) compared to H2O2 (58%). HSE significantly increases catalase activity (31%-50ug versus 51%,-100ug) compared to H2O2 (23%). HSE significantly increases mitochondrial complex 1 activity (125%-50ug versus 229%-100ug) compared to H2O2 (26.2%). There was no significant change in superoxide dismutase activity amongst groups. Conclusion: These observations suggest that HSE creates an antioxidant defense system that provides cytoprotection against H2O2-induced neuronal oxidative stress. Thus, HS, a known anti-hypertensive should be revisited to investigate its in vivo role in neurogenic hypertension.

70 Background: Dysregulation of the HER/EGFR family is identified in esophageal cancers and confers resistance and inferior survival rates. In addition to their unique selectivity in killing DNA repair-deficient tumors, poly-ADP ribose polymerase inhibitors (PARPi) can enhance radiation-induced cytotoxicity. We and others have also previously demonstrated attenuation of DNA repair capacity in HER-inhibited cells to induce a contextual synthetic lethal interaction with PARPi. We thus hypothesized that erlotinib, a tyrosine kinase inhibitor directed against HER1/HER2, could induce a transient DNA repair deficit and subsequently increase DNA damage with the PARPi veliparib in esophageal cancer cells while increasing tumor radiation (RT) sensitivity. Methods: Esophageal SCC cell lines (KYSE-30, KYSE-410, and OE-21) were treated with combinations of vehicle, erlotinib, veliparib (a PARP1/2 inhibitor), and RT. DNA damage and repair and signaling proteins were assessed by immunofluorescence staining of cells for DNA damage and repair foci and/or western blot analysis. Cell viability and cytotoxicity were determined via cell proliferation assays and colony formation assays, respectively. Tumor growth delay was assessed in mice bearing esophageal tumor xenografts. Results: Consistent with our hypotheses, erlotinib increased γ-H2AX foci, a marker of DNA double strand breaks (DSBs), in all three esophageal SCC tumor cells. This coincided with reduced DSB-repair capacity as assessed via RAD51 foci and pDNA-PK. Triple combination with erlotinib, veliparib, and RT demonstrated enhanced cytotoxicity. A subsequent increase in Annexin positive cells was observed, indicative of activation of the apoptotic response. Importantly, the triple combination was most effective in suppressing the growth of esophageal tumor xenografts in vivo (2– to 5.5–fold lower tumor volume) relative to other treatment groups. Furthermore, we observed reductions in tumor volume from baseline with this triple combination in 3/6 mice. Conclusions: Thus, the combination of erlotinib, PARPi, and RT can be an innovative and effective treatment strategy to enhance the therapeutic ratio and improve outcomes in esophageal SCC cancer patients.

Thyroid hormone (TH) receptors (TRs) play central roles in metabolism and are major targets for pharmaceutical intervention. Presently, however, there is limited information about genome wide localizations of TR binding sites. Thus, complexities of TR genomic distribution and links between TRβ binding events and gene regulation are not fully appreciated. Here, we employ a BioChIP approach to capture TR genome-wide binding events in a liver cell line (HepG2). Like other NRs, TRβ appears widely distributed throughout the genome. Nevertheless, there is striking enrichment of TRβ binding sites immediately 5' and 3' of transcribed genes and TRβ can be detected near 50% of T3 induced genes. In contrast, no significant enrichment of TRβ is seen at negatively regulated genes or genes that respond to unliganded TRs in this system. Canonical TRE half-sites are present in more than 90% of TRβ peaks and classical TREs are also greatly enriched, but individual TRE organization appears highly variable with diverse half-site orientation and spacing. There is also significant enrichment of binding sites for TR associated transcription factors, including AP-1 and CTCF, near TR peaks. We conclude that T3-dependent gene induction commonly involves proximal TRβ binding events but that far-distant binding events are needed for T3 induction of some genes and that distinct, indirect, mechanisms are often at play in negative regulation and unliganded TR actions. Better understanding of genomic context of TR binding sites will help us determine why TR regulates genes in different ways and determine possibilities for selective modulation of TR action.

Thyroid hormone (TH) modulates serum cholesterol by acting on TH receptor β1 (TRβ1) in liver to regulate metabolic gene sets. In rodents, one important TH regulated step involves induction of Cyp7a1, an enzyme in the cytochrome P450 family, which enhances cholesterol to bile acid conversion and plays a crucial role in regulation of serum cholesterol levels. Current models suggest, however, that Cyp7a1 has lost the capacity to respond to THs in humans. We were prompted to re-examine TH effects on cholesterol metabolic genes in human liver cells by a recent study of a synthetic TH mimetic which showed that serum cholesterol reductions were accompanied by increases in a marker for bile acid synthesis in humans. Here, we show that TH effects upon cholesterol metabolic genes are almost identical in mouse liver, mouse and human liver primary cells and human hepatocyte cell lines. Moreover, Cyp7a1 is a direct TR target gene that responds to physiologic TR levels through a set of distinct response elements in its promoter. These findings suggest that THs regulate cholesterol to bile acid conversion in similar ways in humans and rodent experimental models and that manipulation of hormone signaling pathways could provide a strategy to enhance Cyp7a1 activity in human patients.