An inverse relationship between protein kinase C (PKC) activity and oestrogen receptor (ER) expression in human breast cell lines and tumours has been firmly established over the past 10 years. To determine whether specific alterations in PKC expression accompany hormone-independence, we examined the expression of PKC isozymes in the hormone-independent human breast cancer cell clones MCF-7 5C and T47D:C42 compared with their hormone-dependent counterparts, MCF-7 A4, MCF-7 WS8 and T47D:A18 respectively. Both hormone-independent cell clones exhibit elevated PKCα expression and increased basal AP-1 activity compared with the hormone-dependent cell clones. To determine whether PKCα overexpression is sufficient to mediate the hormone-independent phenotype, we stably transfected an expression plasmid containing PKCα cDNA to the T47D:A18 and MCF-7 A4 cell lines. This is the first report of PKCα transfection in T47D cells. In contrast to MCF-7 cells, T47D has the propensity to lose the ER and more readily forms tamoxifen-stimulated tumours in athymic mice. We find that in T47D:A18/PKCα clones, there is concomitant up-regulation of PKC βI and δ, whereas in the MCF-7 A4/PKCα transfectants PKC ɛ is up-regulated. In T47D:A18, but not in MCF-7 A4, PKCα stable transfection is accompanied by down-regulation of ER function whilst basal AP-1 activity is elevated. Our results suggest PKCα overexpression may play a role in growth signalling during the shift from hormone dependent to hormone-independent breast cancers. © 2000 Cancer Research Campaign

Estrogen-related receptors (ERR) are orphan members of the nuclear receptor superfamily most closely related to estrogen receptors (ER). Although ERA is a successful target for treating breast cancer, there remains an unmet medical need especially for estrogen-independent breast cancer. Although estradiol is not an ERR ligand, ER and ERR share many commonalities and overlapping signaling pathways. An endogenous ERR ligand has not been identified; however, novel synthetic ERRA subtypespecific antagonists have started to emerge. In particular, we recently identified a novel compound,annulen-5-amine (termed compound A) that acts specifically as an ERRA antagonist. Here, we show that compound A inhibited cell proliferation in ERApositive (MCF-7 and T47D) and ERA-negative (BT-20 and MDA-MD-231) breast cancer cell lines. Furthermore, we report the differential expression of 83 genes involved in ERRA signaling in MCF-7 and BT-20 breast cancer cell lines. We show that compound A slowed tumor growth in MCF-7 and BT-20 mouse xenograft models, and displayed antagonistic effects on the uterus. Furthermore, a subset of genes involved in ERRA signaling in vitro were evaluated and confirmed in vivo by studying uterine gene expression profiles from xenograft mice. These results suggest for the first time that inhibition of ERRA signaling via a subtype-specific antagonist may be an effective therapeutic strategy for ER-positive and ER-negative breast cancers. [Mol Cancer Ther 2009;8(3):672 -81]

IMPORTANCE Because cancer drugs given in combination have the potential for increased tumorcell killing, finding the best combination partners for programmed cell death 1 (PD-1) checkpoint inhibitors could improve clinical outcomes for patients with cancer.OBJECTIVE To identify optimal strategies for combining PD-1 immune checkpoint inhibitors with other cancer therapies. DESIGN, SETTING, AND PARTICIPANTSThis cross-sectional study compiled 319 results from 98 clinical trials testing PD-1 pathway inhibitors alone or in combination with other agents among 24 915 patients with metastatic cancer. All clinical trials had a primary completion EXPOSURES Patients with metastatic cancer were treated with PD-1 immune checkpoint inhibitors alone or with other cancer therapies. MAIN OUTCOMES AND MEASURES Clinical activity was measured as objective response rates (ORRs). Combination measures included fold change from monotherapy to combination ORR, comparison of observed combination ORRs with estimated combination ORRs based on independent additivity, and a computational model to assess clinical synergy. To assess whether theORRs of various combinations may be greater than the independent contribution of each agent, a Bliss independent activity model was used to analyze observed combination ORRs, and a Z score, measuring the difference between observed and calculated ORRs, was generated. RESULTSIn 319 results from 98 clinical trials among 24 915 patients, ORRs for monotherapy were compared with combination data by indication and line of therapy, demonstrating an increased ORR in 105 of 127 results (82.7%) where ORRs were available for both PD-1 pathway inhibitor monotherapy and combination therapy. A few combinations showed increases above the Blissestimated activity, possibly identifying limited clinical synergy. The mean (SD) Z score for all trials was 0.0430 (0.0243). The mean (SD) Z score was 0.0923 (0.0628) for platinum chemotherapy regimen combinations, 0.0547 (0.0821) for vascular endothelial growth factor or vascular endothelial growth factor receptor tyrosine kinase inhibitor combinations, 0.0893 (0.086) for indoleamine 2,3-dioxygenase inhibitor combinations, and 0.0558 (0.0849) for cytotoxic T-lymphocyteassociated protein 4 inhibitor combinations. CONCLUSIONS AND RELEVANCEIn this cross-sectional study, most combination trials showed the expected benefit of combining 2 active anticancer agents, but few combination trials showed clinical synergy according to the Bliss independent activity model. Question What therapies are best combined with programmed cell death 1 (PD-1) checkpoint inhibitors to improve outcomes for patients with cancer who do not respond to PD-1 pathway inhibitor monotherapy? Findings This cross-sectional study of 98 clinical trials, which included 24 915 patients with metastatic cancer, compared objective response rates of PD-1 checkpoint immunotherapies used alone and in combination. Most combinations succeeded, given that the fold change from monotherapy ORR to combination ORR increased in 82.7% ...

Several breast cancer tumor models respond to estradiol (E 2 ) by undergoing apoptosis, a phenomenon known to occur in clinical breast cancer. Before the application of tamoxifen as an endocrine therapy, high-dose E 2 or diethystilbesterol treatment was successfully used, albeit with unfavorable side effects. It is now recognized that such an approach may be a potential endocrine therapy option. We have explored the mechanism of E 2 -induced tumor regression in our T47D:A18/PKCα tumor model that exhibits autonomous growth, tamoxifen resistance, and E 2 -induced tumor regression. Fulvestrant, a selective estrogen receptor (ER) down-regulator, prevents T47D:A18/PKCα E 2 -induced tumor growth inhibition and regression when given before or after tumor establishment, respectively. Interestingly, E 2 -induced growth inhibition is only observed in vivo or when cells are grown in Matrigel but not in two-dimensional tissue culture, suggesting the requirement of the extracellular matrix. Tumor regression is accompanied by increased expression of the proapoptotic FasL/FasL ligand proteins and down-regulation of the prosurvival Akt pathway. Inhibition of colony formation in Matrigel by E 2 is accompanied by increased expression of FasL and short hairpin RNA knockdown partially reverses colony formation inhibition. Classic estrogen-responsive element-regulated transcription of pS2, PR, transforming growth factor-α, C3, and cathepsin D is independent of the inhibitory effects of E 2 . A membrane-impermeable E 2 -BSA conjugate is capable of mediating growth inhibition, suggesting the involvement of a plasma membrane ER. We conclude that E 2 -induced T47D:A18/PKCα tumor regression requires participation of ER-α, the extracellular matrix, FasL/FasL ligand, and Akt pathways, allowing the opportunity to explore new predictive markers and therapeutic targets. (Mol Cancer Res 2009;7(4): 498-510)