The type I transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) is expressed in brain and prostate and overexpressed in prostate cancer, but its role in this disease is unclear. Several studies have suggested that TMEFF2 plays a role in suppressing the growth and invasive potential of human cancer cells, whereas others suggest that the shed portion of TMEFF2, which lacks the cytoplasmic region, has a growth-promoting activity. Here we show that TMEFF2 has a dual mode of action. Ectopic expression of wild-type fulllength TMEFF2 inhibits soft agar colony formation, cellular invasion, and migration and increases cellular sensitivity to apoptosis. However, expression of the ectodomain portion of TMEFF2 increases cell proliferation. Using affinity chromatography and mass spectrometry, we identify sarcosine dehydrogenase (SARDH), the enzyme that converts sarcosine to glycine, as a TMEFF2-interacting protein. Co-immunoprecipitation and immunofluorescence analysis confirms the interaction of SARDH with full-length TMEFF2. The ectodomain does not bind to SARDH. Moreover, expression of the full-length TMEFF2 but not the ectodomain results in a decreased level of sarcosine in the cells. These results suggest that the tumor suppressor activity of TMEFF2 requires the cytoplasmic/transmembrane portion of the protein and correlates with its ability to bind to SARDH and to modulate the level of sarcosine.The transmembrane protein with an epidermal growth factor and two follistatin motifs 2 (TMEFF2) 3 is an evolutionarily conserved type I transmembrane protein expressed in the embryo (1, 2) and selectively in the adult brain and prostate (3-5). The extracellular (ecto-) domain can be cleaved from the membrane in an ADAM17/␥-secretase-dependent fashion (6, 7) and consists of an epidermal growth factor-like motif and two follistatin motifs. The cytoplasmic domain contains a potential G-protein activation motif (2). A critical role for this protein in tumorigenesis is suggested by the fact that it is upregulated in a significant fraction of primary and metastatic prostate tumors (3)(4)(5)8). In fact, ectopic expression or the addition of purified recombinant TMEFF2 ectodomain promotes neuronal cell survival (9), cell growth (6), and phosphorylation of erbB4 and ERK1/2 (2, 6). However, it has also been suggested that TMEFF2 functions as a tumor suppressor because ectopic expression of full-length TMEFF2 demonstrates in vitro antiproliferative effects (4, 10) and suppresses tumor growth in vivo in nude mouse xenografts (10). Consistent with a tumor suppressor activity, Tmeff2 has been shown to be hypermethylated in a number of cancer types (Refs. 11-16 and references therein), and the Tmeff2 promoter is repressed by c-Myc (17).Recently, sarcosine, a glycine derivative, was identified as a potential marker of prostate cancer progression (18). Sarcosine levels were highest in metastatic cancer, and in urine, its levels were higher in men with prostate cancer than in controls. Importantly, using cell ...

BACKGROUND The transmembrane protein with epidermal growth factor and two follistatin motifs, TMEFF2, has been implicated in prostate cancer but its role in this disease is unclear. We recently demonstrated that the tumor suppressor role of TMEFF2 correlates, in part, with its ability to interact with sarcosine dehydrogenase (SARDH) and modulate sarcosine level. TMEFF2 overexpression inhibits sarcosine-induced invasion. Here, we further characterize the functional interaction between TMEFF2 and SARDH and their link with one-carbon (1-C) metabolism and invasion. METHODS RNA interference was used to study the effect of SARDH and/or TMEFF2 knockdown (KD) in invasion, evaluated using Boyden chambers. The dependence of invasion on 1-C metabolism was determined by examining sensitivity to methotrexate. Real-time PCR and western blot of subcellular fractions were used to study the effect of SARDH KD or TMEFF2 KD on expression of enzymes involved in one carbon (1-C) metabolism and on TMEFF2 expression and localization. Protein interactions were analyzed by mass-spectrometry. Cell viability and proliferation were measured by cell counting and MTT analysis. RESULTS While knocking down SARDH affects TMEFF2 subcellular localization, this effect is not responsible for the increased invasion observed in SARDH KD cells. Importantly, SARDH and/or TMEFF2 KD promote increased cellular invasion, sensitize the cell to methotrexate, render the cell resistant to invasion induced by sarcosine, a metabolite from the folate-mediated 1-C metabolism pathway, and affect the expression level of enzymes involved in that pathway. CONCLUSIONS Our findings define a role for TMEFF2 and the folate-mediated 1-C metabolism pathway in modulating cellular invasion.

Results: AA diabetic women, like WA diabetic women, had a larger average very low density lipoprotein (VLDL) size, elevated levels of small low density lipoprotein cholesterol (LDL-C), and lower levels of small high density lipoprotein cholesterol (HDL-C), when compared to lean controls (p Ͻ 0.05). These differences were accompanied by higher VLDL-triglycerides (TG) and LDL-C in WA (p Ͻ 0.05), but not in AA. Although the effects of obesity and diabetes on lipoprotein subpopulation were fairly similar for AA and WA, some racial differences, particularly with respect to HDL, were observed. Discussion:The atherogenic perturbations in lipoprotein profiles of obese AA women, particularly those with diabetes, were relatively similar to those found in WA women and may be contributing to the increased rate of cardiovascular disease (CVD) in AA with obesity and diabetes. The parameters of subpopulation distribution may provide better markers for CVD than lipid concentrations alone, particularly in AA women. Furthermore, subtle racial differences in lipoprotein profiles suggest that race-specific criteria may be needed to screen patients for CVD.

The type I transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2), is expressed mainly in brain and prostate. Expression of TMEFF2 is deregulated in prostate cancer, suggesting a role in this disease, but the molecular mechanism(s) involved in this effect are not clear. Although androgens promote tmeff2 transcription, androgen delivery to castrated animals carrying CWR22 xenografts increases TMEFF2 protein levels in the absence of mRNA changes, suggesting that TMEFF2 may also be post-transcriptionally regulated. Here we show that translation of TMEFF2 is regulated by androgens. Addition of physiological concentrations of dihydrotestosterone (DHT) to prostate cancer cell lines increases translation of endogenous TMEFF2 or transfected TMEFF2-Luciferase fusions, and this effect requires the presence of upstream open reading frames (uORFs) in the 5′-untranslated region (5′-UTR) of TMEFF2. Using chemical and siRNA inhibition of the androgen receptor (AR), we show that the androgen effect on TMEFF2 translation is mediated by the AR. Importantly, DHT also promotes phosphorylation of the α subunit of the translation initiation factor 2 (eIF2α) in an AR-dependent manner, paralleling the effect on TMEFF2 translation. Moreover, endoplasmic reticulum (ER) stress conditions, which promote eIF2α phosphorylation, also stimulate TMEFF2 translation. These results indicate that androgen signaling promotes eIF2α phosphorylation and subsequent translation of TMEFF2 via a mechanism that requires uORFs in the 5′-UTR of TMEFF2.

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