Activated by thyroid hormone, the MAPK (ERK1/2) signaling pathway causes serine phosphorylation by MAPK of several nucleoproteins, including the nuclear thyroid hormone receptor beta1. Because estrogen can activate MAPK and cause MAPK-dependent serine phosphorylation of nuclear estrogen receptor (ER)alpha, we studied whether thyroid hormone also promoted MAPK-mediated ERalpha phosphorylation. Human breast cancer (MCF-7) cells were incubated with physiological concentrations of l-T(4) or 17beta-estradiol (E(2)) for 15 min to 24 h, and nuclear ERalpha and serine-118-phosphorylated ERalpha were identified by Western blotting. Serine-118-phosphorylated ERalpha was recovered at 15 min in nuclei of MCF-7 cells exposed to either T(4) or E(2). The T(4) effect was apparent at 15 min and peaked at 2 h, whereas the E(2) effect was maximal at 4-6 h. T(4)-agarose was as effective as T(4) in causing phosphorylation of ERalpha. T(4) action on ERalpha was inhibited by PD 98059, an inhibitor of ERK1/2 phosphorylation, and by tetraiodothyroacetic acid, a T(4) analog that blocks cell surface-initiated actions of T(4) but is not itself an agonist. Electrophoretic mobility shift assay of nuclear extracts from T(4)-treated and E(2)-treated cells showed similar specific protein-DNA-binding. Indexed by [(3)H]thymidine incorporation and nuclear proliferating cell nuclear antigen, MCF-7 cell proliferation was stimulated by T(4) and T(4)-agarose to an extent comparable with the effect of E(2). This T(4) effect was blocked by either PD 98059 or ICI 182,780, an ER antagonist. Thus, T(4), like E(2), causes phosphorylation by MAPK of nuclear ERalpha at serine-118 in MCF-7 cells and promotes cell proliferation through the ER by a MAPK-dependent pathway.

Recent evidence suggests that the thyroid hormone L-thyroxine (T 4 ) stimulates growth of cancer cells via a plasma membrane receptor on integrin A V B 3 . The contribution of this recently described receptor for thyroid hormone and receptor-based stimulation of cellular mitogen-activated protein kinase [MAPK; extracellular signal-regulated kinase 1/2 (ERK1/2)] activity, to enhancement of cell proliferation by thyroid hormone was quantitated functionally and by immunologic means in three glioma cell lines exposed to T 4 . At concentrations of 1 to 100 nmol/L, T 4 caused proliferation of C6, F98, and GL261 cells, measured by accumulation of proliferating cell nuclear antigen (PCNA) and radiolabeled thymidine incorporation. This effect was inhibited by the T 4 analogue, tetraiodothyroacetic acid, and by an A V B 3 RGD recognition site peptide, both of which block T 4 binding to integrin A V B 3 but are not agonists. Activation of MAPK by T 4 was similarly inhibited by tetraiodothyroacetic acid and the RGD peptide. The thyroid hormone 3,5,3 ¶-triiodo-L-thyronine (T 3 ) and T 4 were equipotent stimulators of PCNA accumulation in C6, F98, and GL261 cells, but physiologic concentrations of T 3 are 50-fold lower than those of T 4 . In conclusion, our studies suggest that glioblastoma cells are thyroid hormone dependent and provide a molecular basis for recent clinical observations that induction of mild hypothyroidism may improve duration of survival in glioblastoma patients. The present experiments infer a novel cell membrane receptor-mediated basis for the growth-promoting activity of thyroid hormone in such tumors and suggest new therapeutic approaches to the treatment of patients with glioblastoma. (Cancer Res 2006; 66(14): 7270-5)

Resveratrol is a naturally occurring polyphenol, which causes apoptosis in cultured cancer cells. We describe a cell surface resveratrol receptor on the extracellular domain of hetero-dimeric alphaVbeta3 integrin in MCF-7 human breast cancer cells. This receptor is linked to induction by resveratrol of extracellular-regulated kinases 1 and 2 (ERK1/2)- and serine-15-p53-dependent phosphorylation leading to apoptosis. The integrin receptor is near the Arg-Gly-Asp (RGD) recognition site on the integrin; an integrin-binding RGD peptide inhibits induction by resveratrol of ERK1/2- and p53-dependent apoptosis. Antibody (Ab) to integrin alphaVbeta3, but not to alphaVbeta5, inhibits activation by resveratrol of ERK1/2 and p53 and consequent apoptosis in estrogen receptor-alpha (ERalpha) positive MCF-7, and ERalpha-negative MDA-MB231 cells. Resveratrol is displaced from the purified integrin by an RGD, but not RGE, peptide, and by alphaVbeta3 integrin-specific Ab. Resveratrol action is blocked by siRNAbeta3, but not by siRNAalphaV. [14C]-Resveratrol binds to commercially purified integrin alphaVbeta3 and to alphaVbeta3 prepared from MCF-7 cells; binding of [14C]-resveratrol to the beta3, but not to the alphaV monomer, is displaced by unlabeled resveratrol. In conclusion, binding of resveratrol to integrin alphaVbeta3, principally to the beta3 monomer, is essential for transduction of the stilbene signal into p53-dependent apoptosis of breast cancer cells.

The PD-1 (programmed death-1)/PD-L1 (PD-ligand 1) checkpoint is a critical regulator of activated T cell-cancer cell interactions, defending tumor cells against immune destruction. Nano-diamino-tetrac (NDAT; Nanotetrac) is an anticancer/anti-angiogenic agent targeted to the thyroid hormone-tetrac receptor on the extracellular domain of integrin αvβ3. NDAT inhibits the cancer cell PI3-K and MAPK signal transduction pathways that are critical to PD-L1 gene expression. We examined actions in vitro of thyroid hormone (l-thyroxine, T) and NDAT on PD-L1 mRNA abundance (qPCR) and PD-L1 protein content in human breast cancer (MDA-MB-231) cells and colon carcinoma (HCT116 and HT-29) cells. In MDA-MB-231 cells, a physiological concentration of T (10M total; 10M free hormone) stimulated PD-L1 gene expression by 38% and increased PD-L1 protein by 2.7-fold (p<0.05, all changes). NDAT (10M) reduced PD-L1 in T-exposed cells by 21% (mRNA) and 39% (protein) (p<0.05, all changes). In HCT116 cells, T enhanced PD-L1 gene expression by 17% and protein content by 24% (p<0.05). NDAT reduced basal PD-L1 mRNA by 35% and protein by 31% and in T-treated cells lowered mRNA by 33% and protein by 66%. In HT-29 cells, T increased PD-L1 mRNA by 62% and protein by 27%. NDAT lowered basal and T-stimulated responses in PD-L1 mRNA and protein by 35-40% (p<0.05). Activation of ERK1/2 was involved in T-induced PD-L1 accumulation. We propose that, by a nongenomic mechanism, endogenous T may clinically support activity of the defensive PD-1/PD-L1 checkpoint in tumor cells. NDAT non-immunologically suppresses basal and T-induced PD-L1 gene expression and protein accumulation in cancer cells.