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SFigure 2: Representative picture of a back tumor, which developed in some control neuron transplanted animals following intravenous injection of MADB106 cells in female F344 rats.

β-Endorphin (BEP)–producing neuron in the hypothalamus plays a key role in bringing the stress axis to a state of homeostasis and maintaining body immune defense system. Long-term delivery of BEP to obtain beneficial effect on chemoprevention is challenging, as the peptides rapidly develop tolerance. Using rats as animal models, we show here that transplantation of BEP neurons into the hypothalamus suppressed carcinogens- and hormone-induced cancers in various tissues and prevented growth and metastasis of established tumors via activation of innate immune functions. In addition, we show that intracerebroventricular administration of nanosphere-attached dibutyryl cyclic adenosine monophosphate (dbcAMP) increased the number of BEP neurons in the hypothalamus, reduced the stress response, enhanced the innate immune function, and prevented tumor cell growth, progression, and metastasis. BEP neuronal supplementation did not produce any deleterious effects on general health but was beneficial in suppressing age-induced alterations in physical activity, metabolic, and immune functions. We conclude that the neuroimmune system has significant control over cancer growth and progression, and that activation of the neuroimmune system via BEP neuronal supplementation/induction may have therapeutic value for cancer prevention and improvement of general health. Cancer Prev Res; 8(1); 56–67. ©2014 AACR.

ERK translocation from cytosol to the ER

SFigure 1: Histopathology of livers from control neurons- or BEP neurons-transplanted groups.

SFigure 3: In vitro release of cAMP at various timepoints from 70 nmol and 350 nmol dbcAMP attached to nanospheres. N =5.

SFigure 4: Representative photomicrograph of a section of rat brain showing fluorescently-labeled-nanosphere attached with cAMP (N-cAMP; blue) in the area of 3rd ventricle.

Supplemental Chemical Methods

Supplementary Figure Legends

Supplementary Figures 1-11

SFigure 5: Effect of nanosphere-delivered dbcAMP (N-cAMP) injection on tyrosine hydroxylase (TH) and corticotropin-releasing hormone (CRH) levels in the hypothalamus.

Supplemental Figures and Table

ERK translocation from ER to the nucleus

Supplementary tables S1-8

Time-lapse imaging of A375(Fucci2)-BR cells treated with the control.

Table S1 Mean density values of crystal violet staining of 16 melanoma cell lines treated with 6-thio-dG and PLX4720. Table S2 RNA-seq data related to A375 cells treated with 6-thio-dG. Table S3 RNA-seq data related to A375 cells treated with BIBR 1532. Table S4 Apoptotic index and ssGSEA scores of 11 gene sets in 18 CCLE melanoma cell lines. Table S5 RPPA data related to A375 cells treated with 6-thio-dG. Table S6 Mean density values of crystal violet staining of 6 therapy-resistant melanoma cell lines treated with 6-thio-dG. Table S7 RPPA data related to the comparison between LOX-IMVI parental and BR cells. Table S8 RPPA data related to LOX-IMVI parental cells treated with 6-thio-dG. Table S9 RPPA data related to LOX-IMVI BR cells treated with 6-thio-dG. Table S10 RPPA data related to LOX-IMVI BR xenografts treated with 6-thio-dG. Table S11 The clinical information from patients with metastatic melanoma treated with immunotherapies.

Purpose: Telomerase promoter mutations are highly prevalent in human tumors including melanoma. A subset of patients with metastatic melanoma often fail multiple therapies, and there is an unmet and urgent need to prolong disease control for those patients.Experimental Design: Numerous preclinical therapy-resistant models of human and mouse melanoma were used to test the efficacy of a telomerase-directed nucleoside, 6-thio-2′-deoxyguanosine (6-thio-dG). Integrated transcriptomics and proteomics approaches were used to identify genes and proteins that were significantly downregulated by 6-thio-dG.Results: We demonstrated the superior efficacy of 6-thio-dG both in vitro and in vivo that results in telomere dysfunction, leading to apoptosis and cell death in various preclinical models of therapy-resistant melanoma cells. 6-thio-dG concomitantly induces telomere dysfunction and inhibits the expression level of AXL.Conclusions: In summary, this study shows that indirectly targeting aberrant telomerase in melanoma cells with 6-thio-dG is a viable therapeutic approach in prolonging disease control and overcoming therapy resistance. Clin Cancer Res; 24(19); 4771–84. ©2018 AACR.See related commentary by Teh and Aplin, p. 4629

Supplementary materials and methods

Supplementary Fig. 1: Related to Fig. 2 (A) The percentage of apoptotic and dead cells indicated as PSVue 643+ cells in each of 12 BRAF-mutant melanoma cell lines treated with PLX4720 or 6-thio-dG at indicated doses for 120 hours. Cells were then harvested and co-stained with PSVue643 and Propidium iodide (PI). The average of 2 biological replicates was plotted. (B) SA-β-gal staining of 4 BRAF-mutant melanoma cell lines treated with 6-thio-dG at 5μM for 9 days. A representative image of 3 biological replicates was shown for each experimental sample. (C and D) Mouse weights of 1205Lu (C) and A375 (D) xenografts in each treatment group were shown. upplementary Fig. 2: Related to Fig. 4 (A) Long-term cell growth assay of 4 melanoma cell lines that acquired resistance to MAPKi treated with 6-thio-dG at indicated doses for 12 days. Cells were then fixed and stained with 3 crystal violet. A representative image of 2 biological replicates was shown for each experimental condition. (B-D) Tumor volumes of WM9 BR (B), UACC-903 BR (C) and A2058 CR (D) xenografts that were treated with the vehicle control and 6-thio-dG at indicated doses. (E) The ssGSEA plot of 11 MSigDB gene sets related to telomere and telomerase that were significantly altered in LOX-IMVI BR cells treated with BIBR 1532 (BIBR) or 6-thio-dG (6dG). (F-I) The heatmaps of RPPA data depicting 30 proteins that were most significantly down-regulated in four representative BR cell lines treated with 6-thio-dG, including A375 BR (F), WM3936-1 (G), 1205Lu BR (H) and WM1552C BR (I). (J) Western blotting of proteins that were downregulated in LOX-IMVI BR cells treated with 6-thio-dG (6dG). (K) The immnohistochemical analysis of AXL was performed in A375 tumors that were treated with the control, 6-thio-dG and Dabrafenib. Supplementary Fig. S3: Related to Fig. 5 (A-C) The heatmaps of two telomere transcriptional gene signatures, two melanoma-specific gene sets and two MAPK pathway-related gene sets in three datasets in which transcriptomes of paired pre- and post-treatment tumor biopsies derived from patients who progressed on MAPKi were profiled. 5 Supplementary Fig. S4: Related to Fig. 5 6 The heatmaps of two telomere transcriptional gene signatures, two melanoma-specific gene sets and two MAPK pathway-related gene sets in paired pre-, on- and post-treatment tumor biopsies derived from 16 patients who were treated with immunotherapies. Supplementary Fig. S5: Related to Fig. 6 (A-E) The heatmaps of RPPA data depicting 30 proteins that were most significantly downregulated in 5 short-term cultures or cell lines derived from immunotherapy-resistant tumors that were treated with 6-thio-dG, including 13-456-3-3 (A), 15-1761-1-2 (B), WM4265-1 (C), WM4265-2 (D) and G43 (E).

Background Opioid and beta-adrenergic receptors are recently shown to cross talk via formation of receptor heterodimers to control the growth and proliferation of breast cancer cells. However, the underlying cell signaling mechanism remained unclear. Methods To determine the effect of the interaction of the two systems in breast cancer, we employed triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468, CRISPR or chemical inhibition or activation of beta-adrenergic receptors (B2AR) and mu-opioid receptors (MOR) gene, and PCR array technology and studied aggressive tumor phenotype and signaling cascades. Results We show here that in triple-negative breast cancer cells, the reduction in expression B2AR and MOR by genetic and pharmacological tools leads to a less aggressive phenotype of triple-negative breast cancer cells in vitro and in animal xenografts. Genomic analysis indicates the glycogen synthase kinase 3 (GSK3) pathway as a possible candidate messenger system involved in B2AR and MOR cross talk. GSK3 inactivation in MDA-MB-231 and MDA-MB-468 cells induced similar phenotypic changes as the inhibition of B2AR and/or MOR, while a GSK3 activation by wortmannin reversed the effects of B2AR and/or MOR knockdown on these cells. GSK3 inactivation also prevents B2AR agonist norepinephrine or MOR agonist DAMGO from affecting MDA-MB-231 and MDA-MB-468 cell proliferation. Conclusions These data confirm a role of B2AR and MOR interaction in the control of breast cancer cell growth and identify a possible role of the GSK3 signaling system in mediation of these two receptors’ cross talk. Screening for ligands targeting B2AR and MOR interaction and/or the GSK3 system may help to identify novel drugs for the prevention of triple-negative breast cancer cell growth and metastasis.

Simple Summary Here, we show that propranolol, a beta blocker used to treat high blood pressure, and naltrexone, an opiate antagonist used for drug and alcohol dependence, when combined, produce marked inhibitory effects on tumor growth and tumor mass while improving the survival rate, increasing NK cell activity, and reducing inflammatory cytokine levels in plasma. These antitumor effects resulted from a reduction in tumor cell proliferation, the induction of cellular apoptosis, and the prevention of epithelial–mesenchymal transition in the tumor. Our data identify a novel treatment with a combination of approved classes of drugs in preclinical breast cancer models. Abstract Cancer progression is known to be promoted by increased body stress caused by elevated beta-adrenergic and opioidergic nervous system activities. The effects of β2-adrenergic blocker propranolol (PRO) and μ-opioid receptor antagonist naltrexone (NTX) were tested using a preclinical model of human breast cancer. These drugs, individually, and more potently when combined, inhibited the cell growth and progression of breast cancer cells in vitro in cultures, and in vivo in rat xenografts. The antitumor activities of these drugs were associated with direct cell intrinsic effects, including increased cell growth arrest, elevated levels of apoptotic proteins, and reduced production of epithelial–mesenchymal transition factors by the tumor cells, as well as effects on innate immune activation and reduced inflammatory cytokine levels in plasma. These data suggest that the combined treatments of PRO and NTX produce impressive antitumor effects in the preclinical breast cancer model, and thereby may provide a new combinatorial treatment strategy with more clinical treatment modalities.

Resistance to BRAF and MEK inhibitors (BRAFi + MEKi) in BRAF-mutant tumors occurs through heterogeneous mechanisms, including ERK reactivation and autophagy. Little is known about the mechanisms by which ERK reactivation or autophagy is induced by BRAFi + MEKi. Here, we report that in BRAF-mutant melanoma cells, BRAFi + MEKi induced SEC61-dependent endoplasmic reticulum (ER) translocation of the MAPK pathway via GRP78 and KSR2. Inhibition of ER translocation prevented ERK reactivation and autophagy. Following ER translocation, ERK exited the ER and was rephosphorylated by PERK. Reactivated ERK phosphorylated ATF4, which activated cytoprotective autophagy. Upregulation of GRP78 and phosphorylation of ATF4 were detected in tumors of patients resistant to BRAFi + MEKi. ER translocation of the MAPK pathway was demonstrated in therapy-resistant patient-derived xenografts. Expression of a dominant-negative ATF4 mutant conferred sensitivity to BRAFi + MEKi in vivo. This mechanism reconciles two major targeted therapy resistance pathways and identifies druggable targets, whose inhibition would likely enhance the response to BRAFi + MEKi. Significance: ERK reactivation and autophagy are considered distinct resistance pathways to BRAF + MEK inhibition (BRAFi + MEKi) in BRAFV600E cancers. Here, we report BRAFi + MEKi–induced ER translocation of the MAPK pathway is necessary for ERK reactivation, which drives autophagy. The ER translocation mechanism is a major druggable driver of resistance to targeted therapy. This article is highlighted in the In This Issue feature, p. 305

Breast cancer is the world’s leading cause of cancer mortality in women worldwide. It has been shown that body stress hormones, such as norepinephrine, and opioid peptides interact to control the growth and progression of this cancer. Opioid and beta-adrenergic receptors are recently shown to cross talk via formation of receptor heterodimers to control cellular functions in various cell systems. However, the underlying cell signaling mechanism remains unclear. To determine the effect of the interaction of the two systems in breast cancer, we employed triple-negative breast cancer (TNBC) cell lines MDA-MB-231 and MDA-MB-468, CRISPR, chemical inhibition or activation and, PCR Array technology to study aggressive tumor phenotype and signaling cascades. We found that knock down of the beta-adrenergic (B2AR) or mu-opioidergic receptor (MOR) system via the CRISPR technique or pharmacological agents induces a less aggressive phenotype in triple negative breast cancer cells both in vitro and in vivo, with the effect being potentiated when both receptor systems were inhibited together. The mRNA expression study using a PCR Array targeting Cancer Stem Cell genes shows that B2AR or MOR activated the glycogen synthase kinase 3 (GSK3) signaling cascade. The GSK3 inactivation in MDA-MB-231 and MDA-MB-468 cells induced similar phenotypic changes as was done by the inhibition of B2AR and/or MOR, while a GSK3 activation by wortmannin reversed the effects of B2AR and/or MOR knockdown on these cells. GSK3 inactivation also prevents B2AR agonist norepinephrine or MOR agonist DAMGO from affecting MDA-MB-231 and MDA-MB-468 cell proliferation. These data identify a role of B2AR and MOR interaction in the control of breast cancer cell growth and establish a possible role of the GSK3 signaling system in mediation of these two receptors’ cross talk. (Supported by NIH grant R01 CA20863201). Citation Format: Bénédicte Rousseau, Sengottuvelan Murugan, Ajay Palagani, Dipak K. Sarkar. Beta-2 adrenergic receptor and mu opioid receptor interact to increase the aggressiveness in triple negative breast cancer cells through activation of GSK3 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1523.

Metastasis is the most deadly aspect of cancer and results from acquired gene regulation abnormalities in tumor cells. Transcriptional regulation is an essential component of controlling of gene function and its failure could contribute to tumor progression and metastasis. During cancer progression, deregulation of oncogenic or tumor suppressive transcription factors, as well as master cell fate regulators, collectively influences multiple steps of the metastasis cascade, including local invasion and dissemination of the tumor to distant organs. Transcription factor PAX3/Pax3, which contributes to diverse cell lineages during embryonic development, plays a major role in tumorigenesis. Mutations in this gene can cause neurodevelopmental disease and the existing literature supports that there is a potential link between aberrant expression of PAX3 genes in adult tissues and a wide variety of cancers. PAX3 function is tissue-specific and could contribute to tumorigenesis either directly as oncogene or as a tumor suppressor by losing its function. In this review, we discuss comprehensively the differential role played by PAX3 in various tissues and how its aberrant expression is implicated in disease development. This review particularly highlights the oncogenic and tumor suppressor role played by PAX3 in different cancers and underlines the importance of precisely identifying tissue-specific role of PAX3 in order to determine its exact role in development of cancer.

Lysosomes serve dual roles in cancer metabolism, executing catabolic programs (i.e., autophagy and macropinocytosis) while promoting mTORC1-dependent anabolism. Antimalarial compounds such as chloroquine or quinacrine have been used as lysosomal inhibitors, but fail to inhibit mTOR signaling. Further, the molecular target of these agents has not been identified. We report a screen of novel dimeric antimalarials that identifies dimeric quinacrines (DQ) as potent anticancer compounds, which concurrently inhibit mTOR and autophagy. Central nitrogen methylation of the DQ linker enhances lysosomal localization and potency. An in situ photoaffinity pulldown identified palmitoyl-protein thioesterase 1 (PPT1) as the molecular target of DQ661. PPT1 inhibition concurrently impairs mTOR and lysosomal catabolism through the rapid accumulation of palmitoylated proteins. DQ661 inhibits the in vivo tumor growth of melanoma, pancreatic cancer, and colorectal cancer mouse models and can be safely combined with chemotherapy. Thus, lysosome-directed PPT1 inhibitors represent a new approach to concurrently targeting mTORC1 and lysosomal catabolism in cancer. Significance: This study identifies chemical features of dimeric compounds that increase their lysosomal specificity, and a new molecular target for these compounds, reclassifying these compounds as targeted therapies. Targeting PPT1 blocks mTOR signaling in a manner distinct from catalytic inhibitors, while concurrently inhibiting autophagy, thereby providing a new strategy for cancer therapy. Cancer Discov; 7(11); 1266–83. ©2017 AACR. See related commentary by Towers and Thorburn, p. 1218. This article is highlighted in the In This Issue feature, p. 1201

In recent years, experimental studies demonstrated negative impacts of impaired body stress response on colonic pathologies. In this study, we tested if reducing body stress response by the use of β-endorphin (BEP) neuronal transplants in the hypothalamus suppresses pre-neoplastic and neoplastic lesions. Colon cancer was induced by injecting 1,2-dimethylhydrazine (DMH) for sixteen weeks in Sprague Dawley rats with BEP neuron transplants or control neuron transplants, and their colonic histopathologies, colon tissue levels of pro-inflammatory cytokines and epithelial-mesenchymal transition (EMT) proteins and splenic levels of cytotoxic proteins were measured. Our results revealed that DMH induced tumors in colon at 100% incidence in control rats but failed to induce colonic tumors in 70% of animal with BEP neuronal transplants. The mean volume of tumor at the colon was smaller in BEP neurons transplanted rats than those in controls. Histopathologies of colon tissues revealed that BEP neurons transplanted animals had lesser tissue lesions such as aberrant crypt foci (ACF) and adenocarcinoma development in the colon than those in control groups. Immunohistochemical and western blot analyses identified reduced expression of Ki-67, TNF-α and NF-κB nuclear translocation in colonic tissues of BEP neurons transplanted rats than those in controls. BEP neurons transplanted rats also showed reduced expressions of transcription factors linked to EMT like Snail, Twist, and N-cadherin, but increased the levels of an epithelial cell marker E-cadherin in colon tissue. Furthermore, splenic NK cells cytolytic proteins such as perforin, granzyme B and IFN-γ levels in BEP neurons transplanted rats were higher than those in control rats. These data suggest that BEP neuron transplants suppress the growth and progression of colonic tumors possibly by decreasing inflammatory mileu and EMT via activation of innate immune responses.

Breast cancer is the world's leading cause of cancer mortality among women. Despite advances in both diagnosis and treatment a large number of women die because of metastatic breast cancer. Research demonstrated a strong link between chronic stress and weakened immune system leaving the body prone to disease like cancer. Indeed, psychological stress has been shown to enhance tumor growth and progression. In addition, stress hormones, such as epinephrine, are implicated in the growth, invasiveness and metastasis of cancer. Therefore, targeting the beta-2 adrenergic receptor (ADRB2) in cancer chemoprevention may offer new promises for the design of therapeutic strategies. For that reason, we determine the cellular processes involved in ADRB2 mediated effects on human triple negative breast cancer cell line MDA-MB-231 in cultures and in xenograft tumors. In order to determine the effects of loss of expression of the gene, we suppressed ADRB2 by knock down, using CRISPR technology or, pharmacologically by employing general beta receptor blocker; propranolol and the beta-2 specific; ICI 188,551. Cell proliferation, migration and invasion assays were performed to define a degree of aggressiveness of the cell phenotype. Western Blot analysis on characteristics proteins of the epithelial-mesenchymal transition was used to further characterize the phenotype. PCR Array expression studies were done to analyze modifications of genes expression profile and signaling cascades in the ADRB2 inhibited cells. We found that an inhibition of ADRB2 receptors reduce proliferation, migration and colony formation of MDA-MB-231 cells. Additionally, ADRB2 knock down increased expression of epithelial proteins while decreased mesenchymal markers. The mRNA expression study targeting cancer stem cell genes showed that the inhibition of ADRB2 significantly modifies signaling pathways involved in cell movement, invasion, migration and proliferation such as Wnt/beta-catenin, ILK and Notch signaling. Within these signaling pathways, the expression of ILK and GSK3 seems to be critical. Indeed, we found that the inhibition of GSK3 by specific inhibitor SD-216763 reduced cell proliferation, migration and colony formation of MDA-MB-231 cells. Also, we found that GSK3 activator, Wortmanin, affected ADRB2 mediated cell proliferation, migration and colony formation of MDA-MB-231 cells. These data demonstrate that GSK3 is a downstream effector of the activation of ADRB2 receptor. Finally, the in vivo study showed a diminution of tumor volume and a better survival when RNU nude rats were treated daily with propanolol compared to control group. Preliminary data suggests propanolol treatment reduces GSK3 level in tumor tissues in association with a decrease in tumor growth. These data provide evidence for a role of ILK-GSK3 signaling in ADRB2 regulated breast tumor growth and aggressiveness. (Supported by NIH grant R01 CA20863201) Citation Format: Benedicte Rousseau, Sengottuvelan Murugan, Ajay Palagani, Ananya Tirupathur, Nupur Kadam, Dipak K. Sarkar. Beta-2 adrenergic receptors role in tumor aggressiveness of triple negative breast cancer [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 6067.

Epigenetics involves multiple processes such as DNA methylation, histone code modifications, and noncoding RNAs to regulate gene expression. In recent years the implications of epigenetic mechanisms have emerged in the field of neuroscience especially in brain development, memory, learning, and various cognition processes. Epigenetics also plays a pivotal role during the aging process of the brain which has led to various age-related neurodegenerative diseases. This manuscript portrays the findings of various epigenetic mechanisms that play a critical role and their implications in aging as well as age-related neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and Huntington's disease.

Purpose: Telomerase promoter mutations are highly prevalent in human tumors including melanoma. A subset of patients with metastatic melanoma often fail multiple therapies, and there is an unmet and urgent need to prolong disease control for those patients. Experimental Design: Numerous preclinical therapy-resistant models of human and mouse melanoma were used to test the efficacy of a telomerase-directed nucleoside, 6-thio-2′-deoxyguanosine (6-thio-dG). Integrated transcriptomics and proteomics approaches were used to identify genes and proteins that were significantly downregulated by 6-thio-dG. Results: We demonstrated the superior efficacy of 6-thio-dG both in vitro and in vivo that results in telomere dysfunction, leading to apoptosis and cell death in various preclinical models of therapy-resistant melanoma cells. 6-thio-dG concomitantly induces telomere dysfunction and inhibits the expression level of AXL. Conclusions: In summary, this study shows that indirectly targeting aberrant telomerase in melanoma cells with 6-thio-dG is a viable therapeutic approach in prolonging disease control and overcoming therapy resistance. Clin Cancer Res; 24(19); 4771–84. ©2018 AACR. See related commentary by Teh and Aplin, p. 4629

Breast cancer is the world’s leading cause of cancer mortality among women. Despite advances in both diagnosis and treatment a still large number of women die because of metastatic breast cancer. Research demonstrated a strong link between chronic stress and weakened immune system leaving the body prone to disease like cancer. Indeed, psychological stress has been shown to enhance tumor growth and progression. In addition, stress hormones, such as norepinephrine and epinephrine, are implicated in the growth, invasiveness and metastasis of cancer. Therefore, targeting the beta-adrenergic receptors in cancer chemoprevention may offer new promises for the design of therapeutic strategies. For that reason, we determine the cellular processes involved in beta-2 adrenergic receptor (ADRB2) mediated effects on human breast cancer cells in cultures. We particularly focused on the impact of the inhibition of ADRB2 on cell proliferation, migration and invasion of the triple negative breast cancer cell line MDA-MB-231. In order to determine the effects of loss of expression of the gene, we suppressed ADRB2 by knock down, using CRISPR technology. We also used pharmacologically suppression of ADBR by employing a general beta receptor blocker; propranolol. Cell proliferation, migration and invasion assays were performed to define a degree of aggressiveness to the cells phenotype after the knock down or pharmacological suppression of ADRB2 in comparison to control cells. Also, Western Blot analysis on characteristics proteins of the epithelial-mesenchymal transition (EMT) was used to further characterize the phenotype. PCR Array expression studies were done to analyze modifications of genes expression profile and signaling cascades in the ADRB2 knocked down cells. We found that the knocking down by CRISPR of the ADRB2 receptors or, pharmacologically suppressing ADRB2 reduces proliferation, migration and colony formation of MDA-MB-231 cells. Additionally, ADRB2 knock down increased expression of epithelial proteins (beta-catenin and E-cadherin) while decreased mesenchymal markers (vimentin and N-cadherin). The mRNA expression study using a PCR Array targeting cancer stem cell genes shows that the inhibition of ADRB2 significantly modifies signaling involved in cell movement, invasion, migration and proliferation such as Wnt/beta-catenin signaling, ILK signaling and Notch signaling. Within these signaling pathways, the expression of several genes such as ILK, MAP2K6, GSK3B and JUN seem to be critical as they are under-expressed. These data provide preliminary understanding of the possible connection between the beta-2 adrenergic receptor system and signaling pathways use by the tumor cells to grow and acquire a more aggressive phenotype. Also, some genes are identified as new targets that may be valuable for developing effective treatment of advanced breast cancer. (Supported by NIH grant R01 CA20863201) Citation Format: Benedicte Rousseau, Ananya Tirupathur, Sengottuvelan Murugan, Dipak K. Sarkar. Beta-2 adrenergic receptors role in tumor aggressiveness of MDA-MB-231 breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3440.

Breast cancer is the second most prevalent cancer after lung cancer among American women. The efficacy of current antitumor therapies is still quite limited. Endogenous opioid peptides, such as endorphin, which is involved in body stress controlling mechanisms, have been shown to enhance immune function and prevent cancer growth and progression in various animal models of cancers. Therefore, targeting the endogenous opioid peptides receptors in the regulation of cancer chemoprevention may offer new promise for the design of therapeutic strategies. Our previous studies have shown differential actions of mu-opioid receptor (MOR) and delta opioid receptor (DOR) on various cell types. In this study we tested the effects of a MOR antagonist naltrexone (NTX) and a DOR agonist (D-Ala2-,N-Me-Phe4,Gly-ol Enkephalin; DPDPE) on proliferation and invasion of human breast cancer cells of different molecular subtype such as T47D (Luminal A), MDA-MB-231 (triple negative, Claudin-Low) and MDA-MB- 468 (triple negative, basal A) in cultures. We used cytotoxicity, clonogenic, cell migration and cell invasion assays to identify effects of these drugs on cancer cells. We found that both NTX and DPDPE have significant cytotoxic effects on these breast cancer cells. Our work from clonogenic, cell migration and cell invasion assays showed that both NTX and DPDPE inhibit the formation of colony, migration and invasion of cancer cells in a concentration-dependent manner. These data suggest that naltrexone and DPDPE have anticancer efficacy in subtypes of human breast cancer cells. The anticancer effects varied depending on the concentration of the drugs and the subtype of breast cancer cells. (Supported by NIH grant R01 CA20863201). Citation Format: Sengottuvelan Murugan, Vanisha Patel, Dipak K. Sarkar. A novel strategy of targeting opioidergic receptors to potentially reduce human breast cancer cell proliferation, colony formation, invasion, and migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3006.

Background Prenatal alcohol exposure has been shown to increase offspring susceptibility to some chemical carcinogens. Whether prenatal exposure to alcohol makes the offspring more susceptible to the development of prostate cancer is not known. Therefore, we determined whether any functional abnormalities and increased cancer susceptibility exist in the prostate of fetal alcohol-exposed male rats during the adult period. Methods Pregnant rats were fed with a liquid diet containing alcohol (alcohol-fed [AF]), or pair-fed with isocaloric liquid diet (PF) or ad libitum fed with rat chow (ad lib-fed). Male offspring of these rats were given N-Nitroso-N-methylurea and testosterone to induce prostate neoplasia or left untreated. Around 6 to 8 months of age, the prostates of these animals were processed for determination of biochemical changes and histopathologies. Results Prostates of noncarcinogen treated animals that were alcohol exposed during the prenatal period demonstrated inflammatory cell infiltration and epithelial atypia and increased number of proliferative cells in the ventral lobe of this gland, but the prostate of control animal showed normal cytoarchitecture. In addition, prenatal alcohol-exposed rats showed decreased levels of cell–cell adhesion marker and increased estrogenic activity in the ventral prostate. Prenatally ethanol (EtOH)-exposed rats, when treated with carcinogen and testosterone, showed histological evidence for high-grade prostatic intraepithelial neoplasia (PIN) primarily in the ventral prostate, whereas control animals showed only low-grade PIN. Prenatally EtOH-exposed rats treated with carcinogen and testosterone also showed increased number of proliferative cells and androgen receptor with concomitant decreased levels of tumor suppressor proteins in the ventral prostate. Conclusions These results suggest for the first time that prenatal EtOH exposures induce histophysiological changes in the prostate as well as it increases the susceptibility of the prostate to develop neoplasia during adulthood.

Epigenetic modifications, including DNA methylation, covalent histone modifications, and small noncoding RNAs, play a key role in regulating the gene expression. This regulatory mechanism is important in cellular differentiation and development. Recent advances in the field of epigenetics extended the role of epigenetic mechanisms in controlling key biological processes such as genome imprinting and X-chromosome inactivation. Aberrant epigenetic modifications are associated with the development of many diseases. The role of epigenetic modifications in various neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, Huntington disease, epilepsy, and multiple sclerosis is rapidly emerging. The use of epigenetic modifying drugs to treat these diseases has been the interest in recent years. A number of natural products having diverse mechanism of action are used for drug discovery. For many years, natural compounds have been used to treat various neurodegenerative diseases, but the use of such compounds as epigenetic modulators to reverse or treat neurological diseases are not well studied. In this chapter, we mainly focus on how various epigenetic modifications play a key role in neurodegenerative diseases, their mechanism of action, and how it acts as a potential therapeutic target for epigenetic drugs to treat these diseases will be discussed.

Epigenetic modifications, including DNA methylation, covalent histone modifications, and small noncoding RNAs, play a key role in regulating the gene expression. This regulatory mechanism is important in cellular differentiation and development. Recent advances in the field of epigenetics extended the role of epigenetic mechanisms in controlling key biological processes such as genome imprinting and X-chromosome inactivation. Aberrant epigenetic modifications are associated with the development of many diseases. The role of epigenetic modifications in various neurodegenerative disorders including Alzheimer’s disease, Parkinson’s disease, Huntington disease, epilepsy, and multiple sclerosis is rapidly emerging. The use of epigenetic modifying drugs to treat these diseases has been the interest in recent years. A number of natural products having diverse mechanism of action are used for drug discovery. For many years, natural compounds have been used to treat various neurodegenerative diseases, but the use of such compounds as epigenetic modulators to reverse or treat neurological diseases are not well studied. In this chapter, we mainly focus on how various epigenetic modifications play a key role in neurodegenerative diseases, their mechanism of action, and how it acts as a potential therapeutic target for epigenetic drugs to treat these diseases will be discussed.

Defective autophagy has been linked with many pathologies, including cancer, diabetes, infectious disease, myopathies, heart, liver, lung, and neurodegenerative disease. Autophagy has therefore become an important target in drug discovery. Recent advances have identified new ways to monitor autophagy in vitro and in vivo. Many assays rely on visualizing autophagy-related intracellular markers such as microtubule-associated protein light chain 3 (LC3) II, which have posed issues with in vivo and clinical translation of the in vitro assays. Here, we present an overview of current in vitro and in vivo methodologies to measure autophagy with a special focus on the tumor microenvironment.

Contains fulltext : 80086.pdf (Publisher’s version ) (Closed access) Cell surface heparan sulfate plays a critical role in regulating the metastatic behavior of tumor cells, whereas the role of chondroitin sulfate/dermatan sulfate (CS/DS) has been little understood in this context. Here, we characterized CS/DS chains from the murine osteosarcoma cell line LM8G7, which forms tumor nodules in liver. Structural analysis of the CS/DS chains showed a higher proportion of GlcUA beta 1-3GalNAc(4,6-O-disulfate) (E-units) in LM8G7 (12%) than in its parental cell line LM8 (6%), which rarely forms tumors in the liver. Immunostaining with GD3G7, an antibody specific to E-units, confirmed the higher expression of the epitope in LM8G7 than LM8 cells. The tumor focal formation of LM8G7 cells in the liver in mice was effectively inhibited by the preadministration of CS-E (rich in E-unit) or the preincubation of the antibody GD3G7 with the tumor cells. CS-E or GD3G7 inhibited the adhesion of LM8G7 cells to a laminin-coated plate in vitro. In addition, the invasive ability of LM8G7 cells in vitro was also reduced by the addition of CS-E or the antibody. Further, CS-E or the antibody inhibited the proliferation of LM8G7 cells dose dependently. The binding of LM8G7 cells to VEGF in vitro was also significantly reduced by CS-E and GD3G7. Thus, the present study reveals the significance of highly sulfated CS/DS structures in the liver colonization of osteosarcoma cells and also provides a framework for the development of GAG-based anticancer molecules.

Contains fulltext : 69607.pdf (Publisher’s version ) (Open Access) The altered expression of cell surface chondroitin sulfate (CS) and dermatan sulfate (DS) in cancer cells has been demonstrated to play a key role in malignant transformation and tumor metastasis. However, the functional highly sulfated structures in CS/DS chains and their involvement in the process have not been well documented. In the present study, a structural analysis of CS/DS from two mouse Lewis lung carcinoma (3LL)-derived cell lines with different metastatic potentials revealed a higher proportion of Delta(4,5)HexUA-GalNAc(4,6-O-disulfate) generated from E-units (GlcUA-GalNAc(4, 6-O-disulfate)) in highly metastatic LM66-H11 cells than in low metastatic P29 cells, although much less CS/DS is expressed by LM66-H11 than P29 cells. This key finding prompted us to study the role of CS-E-like structures in experimental lung metastasis. The metastasis of LM66-H11 cells to lungs was effectively inhibited by enzymatic removal of tumor cell surface CS or by preadministration of CS-E rich in E-units in a dose-dependent manner. In addition, immunocytochemical analysis showed that LM66-H11 rather than P29 cells expressed more strongly the CS-E epitope, which was specifically recognized by the phage display antibody GD3G7. More importantly, this antibody and a CS-E decasaccharide fraction, the minimal structure recognized by GD3G7, strongly inhibited the metastasis of LM66-H11 cells probably by modifying the proliferative and invading behavior of the metastatic tumor cells. These results suggest that the E-unit-containing epitopes are involved in the metastatic process and a potential target for the diagnosis and treatment of malignant tumors.

Background Recently, retrograde tracing has provided evidence for an influence of hypothalamic β-endorphin (BEP) neurons on the liver, but functions of these neurons are not known. We evaluated the effect of BEP neuronal activation on alcohol-induced liver injury and hepatocellular cancer. Methods Male rats received either BEP neuron transplants or control transplants in the hypothalamus and were randomly assigned to feeding alcohol-containing liquid diet or control liquid diet for 8 weeks or to treatment of a carcinogen diethylnitrosamine (DEN). Liver tissues of these animals were analyzed histochemically and biochemically for tissue injuries or cancer. Results Alcohol feeding increased liver weight and induced several histopathological changes such as prominent microvesicular steatosis and hepatic fibrosis. Alcohol feeding also increased the levels of triglyceride, hepatic stellate cell (HSC) activation factors, and catecholamines in the liver and endotoxin levels in the plasma. However, these effects of alcohol on the liver were reduced in animals with BEP neuron transplants. BEP neuron transplants also suppressed carcinogen-induced liver histopathologies such as extensive fibrosis, large focus of inflammatory infiltration, hepatocellular carcinoma (HCC), collagen deposition, numbers of preneoplastic foci, levels of HSC activation factors and catecholamines, as well as inflammatory milieu and increased the levels of natural killer cell cytotoxic factors in the liver. Conclusions These findings are the first evidence for a role of hypothalamic BEP neurons in influencing liver functions. Additionally, the data identify that BEP neuron transplantation prevents hepatocellular injury and HCC formation possibly via influencing the immune function.

Early-life ethanol feeding (ELAF) alters the metabolic function of proopiomelanocortin (POMC)-producing neurons and the circadian expression of clock regulatory genes in the hypothalamus. We investigated whether the circadian mechanisms control the action of ELAF on metabolic signaling genes in POMC neurons. Gene expression measurements of Pomc and a selected group of metabolic signaling genes, Stat3, Sirt1, Pgc1-α, and Asb4 in laser-captured microdissected POMC neurons in the hypothalamus of POMC-enhanced green fluorescent protein mice showed circadian oscillations under light/dark and constant darkness conditions. Ethanol programmed these neurons such that the adult expression of Pomc, Stat3, Sirt, and Asb4 gene transcripts became arrhythmic. In addition, ELAF dampened the circadian peak of gene expression of Bmal1, Per1, and Per2 in POMC neurons. We crossed Per2 mutant mice with transgenic POMC-enhanced green fluorescent protein mice to determine the role of circadian mechanism in ELAF-altered metabolic signaling in POMC neurons. We found that ELAF failed to alter arrhythmic expression of most circadian genes, with the exception of the Bmal1 gene and metabolic signaling regulating genes in Per2 mutant mice. Comparison of the ELAF effects on the circadian blood glucose in wild-type and Per2 mutant mice revealed that ELAF dampened the circadian peak of glucose, whereas the Per2 mutation shifted the circadian cycle and prevented the ELAF dampening of the glucose peak. These data suggest the possibility that the Per2 gene mutation may regulate the ethanol actions on Pomc and the metabolic signaling genes in POMC neurons in the hypothalamus by blocking circadian mechanisms.

The safety and preliminary activity of hydroxychloroquine in phase I cancer clinical trials have established the feasibility and rationale for targeting the lysosome in cancer. We previously reported a more potent lysosomal inhibitor Lys05, which is a dimeric chloroquine (CQ) linked with a triamine linker. Here we report that high throughput screening of >100 Lys05 derivatives (72-hour viability assay) revealed extending linker length between the CQ motifs markedly enhanced anti-proliferative potency (IC50 Citation Format: Vito W. Rebecca, Michael Nicastri, Noel McGlaughlin, Quentin McAfee, Gao Zhang, Gretchen M. Alicea, Shengfu Piao, Colin Fennelly, Sengottuvelan Murugan, Zhi Wei, Gordon B. Mills, Yiling Lu, Meenhard Herlyn, Jeffrey D. Winkler, Ravi K. Amaravadi. Structural features of novel dimeric quinacrines that have single-agent antitumor activity determine the mechanism of action: destabilization of mTORC1/lysosomal interaction versus DNA damage. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1018.