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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.

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.