Resistance to endocrine therapy occurs in virtually all patients with estrogen receptor a (ERa)-positive metastatic breast cancer, and is attributed to various mechanisms including loss of ERa expression, altered activity of coregulators, and cross-talk between the ERa and growth factor signaling pathways. To our knowledge, acquired mutations of the ERa have not been described as mediating endocrine resistance. Samples of 13 patients with metastatic breast cancer were analyzed for mutations in cancer-related genes. In five patients who developed resistance to hormonal therapy, a mutation of A to G at position 1,613 of ERa, resulting in a substitution of aspartic acid at position 538 to glycine (D538G), was identified in liver metastases. Importantly, the mutation was not detected in the primary tumors obtained prior to endocrine treatment. Structural modeling indicated that D538G substitution leads to a conformational change in the ligand-binding domain, which mimics the conformation of activated ligand-bound receptor and alters binding of tamoxifen. Indeed, experiments in breast cancer cells indicated constitutive, ligand-independent transcriptional activity of the D538G receptor, and overexpression of it enhanced proliferation and conferred resistance to tamoxifen. These data indicate a novel mechanism of acquired endocrine resistance in breast cancer. Further studies are needed to assess the frequency of D538G-ERa among patients with breast cancer and explore ways to inhibit its activity and restore endocrine sensitivity. Cancer Res; 73(23); 6856-64. Ó2013 AACR.
Klotho is an anti-aging transmembrane protein, which can be shed and function as a hormone. Accumulating data indicate klotho as a tumor suppressor in a wide array of malignancies and indicate the subdomain KL1 as the active region of the protein. We aimed to study the role of klotho as a tumor suppressor in colorectal cancer. Bioinformatics analyses of TCGA datasets indicated reduced klotho mRNA levels in human colorectal cancer, along with negative regulation of klotho expression by hypermethylation of the promoter and 1st exon, and hypomethylation of an area within the gene. Overexpression or treatment with klotho or KL1 inhibited proliferation of colorectal cancer cells in vitro. The in vivo activity of klotho and KL1 was examined using two models recapitulating development of tumors in the normal colonic environment of immune-competent mice. Treatment with klotho inhibited formation of colon polyps induced by the carcinogen azoxymethane, and KL1 treatment slowed growth of orthotopically-implanted colorectal tumors. Gene expression array revealed that klotho and KL1 expression enhanced the unfolded protein response (UPR) and this was further established by increased levels of spliced XBP1, GRP78 and phosphorylated-eIF2α. Furthermore, attenuation of the UPR partially abrogated klotho tumor suppressor activity. In conclusion, this study indicates klotho as a tumor suppressor in colorectal cancer and identifies, for the first time, the UPR as a pathway mediating klotho activities in cancer. These data suggest that administration of exogenous klotho or KL1 may serve as a novel strategy for prevention and treatment of colorectal cancer.
Klotho is a transmembrane protein containing two internal repeats, KL1 and KL2, both displaying significant homology to members of the b-glycosidase family. Klotho is expressed in the kidney, brain, and various endocrine tissues, but can also be cleaved and act as a circulating hormone. Klotho is an essential cofactor for binding of fibroblast growth factor 23 (FGF23) to the FGF receptor and can also inhibit the insulin-like growth factor-1 (IGF-1) pathway. Data from a wide array of malignancies indicate klotho as a tumor suppressor; however, the structure-function relationships governing its tumor suppressor activities have not been deciphered. Here, the tumor suppressor activities of the KL1 and KL2 domains were examined. Overexpression of either klotho or KL1, but not of KL2, inhibited colony formation by MCF-7 and MDA-MB-231 cells. Moreover, in vivo administration of KL1 was not only well tolerated but significantly slowed tumor formation in nude mice. Further studies indicated that KL1, but not KL2, interacted with the IGF-1R and inhibited the IGF-1 pathway. Based on computerized structural modeling, klotho constructs were generated in which critical amino acids have been mutated. Interestingly, the mutated proteins retained their tumor suppressor activity but showed reduced ability to modulate FGF23 signaling. These data indicate differential activity of the klotho domains, KL1 and KL2, in breast cancer and reveal that the tumor suppressor activities of klotho can be dissected from its physiologic activities.Implications: These findings pave the way for a rational design of safe klotho-based molecules for the treatment of breast cancer.
Purpose: Mutations in the ligand-binding domain (LBD) of estrogen receptor a (ER) confer constitutive transcriptional activity and resistance to endocrine therapies in patients with breast cancer. Accumulating clinical data suggest adverse outcome for patients harboring tumors expressing these mutations. We aimed to elucidate mechanisms conferring this aggressive phenotype.Experimental Design: Cells constitutively expressing physiologic levels of ER-harboring activating LBD mutations were generated and characterized for viability, invasiveness, and tumor formation in vivo. Gene expression profile was studied using microarray and RNAseq technologies. Metabolic properties of the cells were assessed using global metabolite screen and direct measurement of metabolic activity.Results: Cells expressing mutated ER showed increased proliferation, migration, and in vivo tumorigenicity compared with cells expressing the wild-type ER (WT-ER), even in the presence of estrogen. Expression of the mutated ER was associated with upregulation of genes involved in invasion and metastases, as well as elevation of genes associated with tumor cell metabolism. Indeed, a metabolic examination revealed four distinct metabolic profiles: WT-ER-expressing cells either untreated or estrogen treated and mutated ER-expressing cells either untreated or estrogen treated. Pathway analyses indicated elevated tricarboxylic acid cycle activity of 537S-ERexpressing cells. Thus, while WT-ER cells were mostly glucosedependent, 537S-ER were not addicted to glucose and were able to utilize glutamine as an alternative carbon source.Conclusions: Taken together, these data indicate estrogenindependent rewiring of breast cancer cell metabolism by LBD-activating mutations. These unique metabolic activities may serve as a potential vulnerability and aid in the development of novel treatment strategies to overcome endocrine resistance.
Background Estrogen receptor α (ESR1) plays a critical role in promoting growth of various cancers. Yet, its role in the development of pancreatic cancer is not well-defined. A less studied region of ESR1 is the hinge region, connecting the ligand binding and DNA domains. rs142712646 is a rare SNP in ESR1, which leads to a substitution of arginine to cysteine at amino acid 269 (R269C). The mutation is positioned in the hinge region of ESR1, hence may affect the receptor structure and function. We aimed to characterize the activity of R269C-ESR1 and study its role in the development of pancreatic cancer. Methods Transcriptional activity was evaluated by E2-response element (ERE) and AP1 –luciferase reporter assays and qRT-PCR. Proliferation and migration were assessed using MTT and wound healing assays. Gene-expression analysis was performed using RNAseq. Results We examined the presence of this SNP in various malignancies, using the entire database of FoundationOne and noted enrichment of it in a subset of pancreatic non-ductal adenocarcinoma (n = 2800) compared to pancreatic ductal adenocarcinoma (PDAC) as well as other tumor types (0.53% vs 0.29%, p = 0.02). Studies in breast and pancreatic cancer cells indicated cell type-dependent activity of ESR1 harboring R269C. Thus, expression of R269C-ESR1 enhanced proliferation and migration of PANC-1 and COLO-357 pancreatic cancer cells but not of MCF-7 breast cancer cells. Moreover, R269C-ESR1 enhanced E2-response elements (ERE) and AP1-dependent transcriptional activity and increased mRNA levels of ERE and AP1-regulated genes in pancreatic cancer cell lines, but had a modest effect on MCF-7 breast cancer cells. Accordingly, whole transcriptome analysis indicated alterations of genes associated with tumorigenicity in pancreatic cancer cells and upregulation of genes associated with cell metabolism and hormone biosynthesis in breast cancer cells. Conclusions Our study shed new light on the role of the hinge region in regulating transcriptional activity of the ER and indicates cell-type specific activity, namely increased activity in pancreatic cancer cells but reduced activity in breast cancer cells. While rare, the presence of rs142712646 may serve as a novel genetic risk factor, and a possible target for therapy in a subset of non-ductal pancreatic cancers.
<p>S1: LBD-ER mutations are expressed at physiologic levels and are E2-independent. S2: LBD-ER lead to an enrichment pathway of ECM degradation S3: High concentrations of E2 do not recapitulate the mutant phenotype in WT-ER expressing cells. S4: LBD-mutations activate the AKT/mTOR pathway. S5: Mitochondrial activity of D538G-ER expressing T47D cells. S6: Glutamine leads to enrichment of specific pathways by 537S-ER.</p>
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