N-methyladenosine (mA) messenger RNA methylation is a gene regulatory mechanism affecting cell differentiation and proliferation in development and cancer. To study the roles of mA mRNA methylation in cell proliferation and tumorigenicity, we investigated human endometrial cancer in which a hotspot R298P mutation is present in a key component of the methyltransferase complex (METTL14). We found that about 70% of endometrial tumours exhibit reductions in mA methylation that are probably due to either this METTL14 mutation or reduced expression of METTL3, another component of the methyltransferase complex. These changes lead to increased proliferation and tumorigenicity of endometrial cancer cells, likely through activation of the AKT pathway. Reductions in mA methylation lead to decreased expression of the negative AKT regulator PHLPP2 and increased expression of the positive AKT regulator mTORC2. Together, these results reveal reduced mA mRNA methylation as an oncogenic mechanism in endometrial cancer and identify mA methylation as a regulator of AKT signalling.

CDC37, an essential gene in Saccharomyces cerevisiae, interacts genetically with multiple protein kinases and is required for production of Cdc28p/cyclin complexes through an unknown mechanism. We have identified mammalian p50Cdc37 as a protein kinase-targeting subunit of the molecular chaperone Hsp90. Previously, p50 was observed in complexes with pp60v-src and Raf-1, but its identity and function have remained elusive. In mouse fibroblasts, a primary target of Cdc37 is Cdk4. This kinase is activated by D-type cyclins and functions in passage through G1. In insect cells, Cdc37 is sufficient to target Hsp90 to Cdk4 and both in vitro and in vivo, Cdc37/Hsp90 associates preferentially with the fraction of Cdk4 not bound to D-type cyclins. Cdc37 is coexpressed with cyclin Dl in cells undergoing programmed proliferation in vivo, consistent with a positive role in cell cycle progression. Pharmacological inactivation of Cdc37/Hsp90 function decreases the half-life of newly synthesized Cdk4, indicating a role for Cdc37/Hsp90 in Cdk4 stabilization. This study suggests a general role for p50Cdc37 in signaling pathways dependent on intrinsically unstable protein kinases and reveals a previously unrecognized chaperone-dependent step in the production of Cdk4/cyclin D complexes.

Mesenchymal stem cells derived from bone marrow have recently been described to localize to breast carcinomas and to integrate into the tumor-associated stroma. In the present study, we investigated whether adipose tissue-derived stem cells (ASCs) could play a role in tumor growth and invasion. Compared with bone marrow-derived cells, ASCs as tissue-resident stem cells are locally adjacent to breast cancer cells and may interact with tumor cells directly. Here, we demonstrate that ASCs cause the cancer to grow significantly faster when added to a murine breast cancer 4T1 cell line. We further show that breast cancer cells enhance the secretion of stromal cell-derived factor-1 from ASCs, which then acts in a paracrine fashion on the cancer cells to enhance their motility, invasion and metastasis. The tumor-promoting effect of ASCs was abolished by knockdown of the chemokine C-X-C receptor 4 in 4T1 tumor cells. We demonstrated that ASCs home to tumor site and promote tumor growth not only when co-injected locally but also when injected intravenously. Furthermore, we demonstrated that ASCs incorporate into tumor vessels and differentiate into endothelial cells. The tumor-promoting effect of tissue-resident stem cells was also tested and validated using a human breast cancer line MDA-MB-231 cells and human adipose tissue-derived stem cells. Our findings indicate that the interaction of local tissue-resident stem cells with tumor stem cells plays an important role in tumor growth and metastasis.

The C terminus of nuclear hormone receptors is a complex structure that contains multiple functions. We are interested in the mechanism by which thyroid hormone converts its receptor from a transcriptional silencer to an activator of transcription. Both regulatory functions are localized in the ligand binding domain of this receptor superfamily member. In this study, we have identified and characterized several functional domains within the ligand binding domain of the human thyroid hormone receptor (TR␤) conferring transactivation. Interestingly, these domains are localized adjacent to hormone binding sites. One activation domain, designated 4, is only 17 amino acids in length and is localized at the extreme C terminus of TR. Deletion of six amino acids of 4 resulted in a receptor that could still bind hormone but acted as a constitutive silencer, indicating that 4 is required for both transactivation and relief of the silencing functions. In addition, we performed in vivo competition experiments, the results of which suggest that in the absence of 4 or hormone, TR is bound by a corepressor protein(s) and that one role of hormone is to release corepressor from the receptor. We propose a general model in which the role of hormone is to induce a conformational change in the receptor that subsequently affects the action of 4, leading to both relief of silencing and transcriptional activation.The thyroid hormone receptor (TR) belongs to the superfamily of nuclear hormone receptors which includes receptors for steroids, retinoids, and vitamin D 3 . These receptors act as transcription factors which activate gene expression in a ligand-dependent manner. Unlike receptors for the classical steroid hormones, TR is not associated with heat shock proteins in the absence of hormone but is bound to its response element and actively represses promoter activity through a mechanism termed silencing (27, 32). Like other nuclear hormone receptors, TR activates expression of target genes in the presence of hormone (5, 12).Nuclear hormone receptors, like other transcription factors, are composed of modular functional domains which can act as functional units outside of their natural context. As an example, the entire C terminus of the glucocorticoid receptor was transferred to its amino terminus without affecting its transcriptional properties (31). Such an approach also led to the identification of the transactivation domains 1 in the glucocorticoid receptor N terminus and 2, localized C terminal of the DNA binding domain (DBD) (24). The amino termini of some receptors contain an activation domain, which retains normal function when linked to a heterologous DBD such as that of the yeast transcription factor GAL4 (7,24,36). Similarly, the entire C termini of various receptors have been fused to the GAL4 DBD, resulting in hormone-dependent transcription factors (4, 7, 24, 43). GAL-receptor fusions act similarly to wild-type receptors when bound to a GAL4 DNA-binding site. The structures of various other transcription factors have b...

Lipocalin 2 (LCN2; also known as NGAL) is a secreted glycoprotein and its elevated expression has been observed in breast cancers. However, the importance of LCN2 in breast tumorigenesis is unclear. Here, we employed a spontaneous mammary tumor mouse model showing that MMTV-ErbB2 (V664E) mice lacking mouse LCN2 had significantly delayed mammary tumor formation and metastasis with reduced matrix metalloproteinase-9 activity in the blood. LCN2 expression is upregulated by HER2/phosphoinositide 3-kinase/ AKT/NF-κB pathway. Decreasing LCN2 expression significantly reduced the invasion and migration ability of HER2 + breast cancer cells. Furthermore, injecting an anti-mouse LCN2 antibody into mice bearing established murine breast tumors resulted in significant blockage of lung metastasis. Our findings indicate that LCN2 is a critical factor in enhancing breast tumor formation and progression possibly in part by stabilizing matrix metalloproteinase-9. Our results suggest that inhibition of LCN2 function by an inhibitory monoclonal antibody has potential for breast cancer therapy, particularly by interfering with metastasis in aggressive types of breast cancer.