Follistatin-like 3 (FSTL3) binds and inactivates activin, a growth factor involved with cell growth and differentiation. We have previously shown FSTL3 overexpression in invasive breast cancers, but its clinical relevance remained unexplored. Here we evaluate FSTL3 as a prognostic tool and its relation with clinical and pathological features of breast cancer. A cohort of 154 women diagnosed with invasive breast cancer between 2008 and 2012 was followed up for 5 years. Tumor samples were processed by immunohistochemistry to detect FSTL3 expression in tumor epithelium. FSTL3 expression was classified semiquantitatively and tested for possible correlation with age, menopause status, stage, tumor histological type and grade, estrogen receptor, progesterone receptor, and HER2 expression. Survival plots with Kaplan-Mayer statistics were used to assess whether FSTL3 expression predicted disease-free survival. Our findings show that FSTL3 staining was unrelated to menopausal status, histological type, disease stage, or receptor profile. However, the intensity of FSTL3 immunostaining correlated inversely with tumor size (r = -0.366, p<0.001) and with nuclear grade (p<0.01). The intensity of FSTL3 expression in the tumoral epithelium was not predictive of the disease-free survival (p = 0.991, log-rank test), even though the follow-up length and the study size were sufficient to detect a significant reduction in disease-free survival among women with stage III-IV compared to stage I-II disease (p<0.001). FSTL3 expression in invasive breast cancer is inversely associated with tumor size and nuclear grade but it does not predict disease relapse in the short term.

Breast cancer is a heterogeneous disease whose molecular diversity is not well reflected in clinical and pathological markers used for prognosis and treatment selection. As tumor cells secrete proteins into the extracellular environment, some of these proteins reach circulation and could become suitable biomarkers for improving diagnosis or monitoring response to treatment. As many signaling pathways and interaction networks are altered in cancerous tissues by protein phosphorylation, changes in the secretory phosphoproteome of cancer tissues could reflect both disease progression and subtype. To test this hypothesis, we compared the phosphopeptide-enriched fractions obtained from proteins secreted into conditioned media (CM) derived from five luminal and five basal type breast cancer cell lines using label-free quantitative mass spectrometry. Altogether over 5000 phosphosites derived from 1756 phosphoproteins were identified, several of which have the potential to qualify as phosphopeptide plasma biomarker candidates for the more aggressive basal and also the luminal-type breast cancers. The analysis of phosphopeptides from breast cancer patient plasma and controls allowed us to construct a discovery list of phosphosites under rigorous collection conditions, and second to qualify discovery candidates generated from the CM studies. Indeed, a set of basal-specific phosphorylation CM site candidates derived from IBP3, CD44, OPN, FSTL3, LAMB1, and STC2, and luminal-specific candidates derived from CYTC and IBP5 were selected and, based on their presence in plasma, quantified across all cell line CM samples using Skyline MS1 intensity data. Together, this approach allowed us to assemble a set of novel cancer subtype specific phosphopeptide candidates for subsequent biomarker verification and clinical validation.

The cancer invasion front (CIF), a spatially-recognized area due to the frequent presence of peritumoral desmoplastic reaction, represents a cancer site where many hallmarks of cancer metastasis occur. It is now strongly suggested that the desmoplastic microenvironment holds crucial information for determining tumor development and progression. Despite extensive research on tumor-host cell interactions at CIFs, the exact paracrine molecular network that is hardwired into the proteome of the stromal and cancer subpopulations remains partially understood. Here, we interrogated the signaling pathways and the molecular functional signatures across the proteome of a desmoplastic coculture model system of colorectal cancer progression. We discovered a group of bone morphogenetic protein (BMP) antagonists that coordinates major biological programs in CIFs, including cell proliferation, invasion, migration and differentiation processes. Using a mathematical model of cancer cell progression, coupled to in vitro cell migration assays, we demonstrated that the prominent BMP antagonist gremlin-1 (GREM1) may trigger motility of cancer cell cohorts. Our data collectively demonstrate that the desmoplastic CIFs deploy a microenvironmental signature, based on BMP antagonism, in order to regulate the motogenic fates of cancer cell cohorts invading the adjacent stroma.

CONTEXT: Investigation of activin-A (A) and myostatin (M) in human myometrium (HM) and leiomyoma (HL) will explain their involvement in human myometrial pathophysiology.
OBJECTIVE: We aimed to investigate A and M response and steroid regulation in HM. We also evaluated A and M expression and response in HL.
DESIGN: Tissues were analyzed and cultured.
PATIENTS: Patients included fertile (in proliferative phase) and menopausal women undergoing hysterectomy.
INTERVENTIONS: HM explant cultures were treated with A and M (for Smad-7 mRNA quantification) or estrogen and progesterone (for A and M mRNA quantification). A and M expression levels were also evaluated in menopausal (physiological absence of steroids) HM specimens. A and M and their receptors were evaluated in HL (n = 8, diameter 5-8 cm) compared with their matched HM. HL explants cultures were treated with A and M (for Smad7 mRNA quantification), and, to explain the absence of response, the levels of follistatin, follistatin-related gene (FLRG), and Cripto were evaluated.
RESULTS: A and M increased Smad7 expression in HM explants. A and M mRNAs were both reduced after estradiol treatment, unchanged after progesterone treatment, but were higher in menopausal than fertile (in proliferative phase) specimens. A, M, and FLRG were expressed at higher levels in HL compared with adjacent HM, whereas the receptors, follistatin, and Smad7 mRNAs resulted unchanged. Cripto mRNA was expressed only in HL.
CONCLUSIONS: A and M act on human HM and are regulated by steroids. In HL there is an increase of A, M, FLRG, and Cripto expression.

BACKGROUND: Activins are growth factors acting on cell growth and differentiation. Activins are expressed in high grade breast tumors and they display an antiproliferative effect inducing G0/G1 cell cycle arrest in breast cancer cell lines. Follistatin and follistatin- related gene (FLRG) bind and neutralize activins. In order to establish if these activin binding proteins are involved in breast tumor progression, the present study evaluated follistatin and FLRG pattern of mRNA and protein expression in normal human breast tissue and in different breast proliferative diseases.
METHODS: Paraffin embedded specimens of normal breast (NB - n = 8); florid hyperplasia without atypia (FH - n = 17); fibroadenoma (FIB - n = 17); ductal carcinoma in situ (DCIS - n = 10) and infiltrating ductal carcinoma (IDC - n = 15) were processed for follistatin and FLRG immunohistochemistry and in situ hybridization. The area and intensity of chromogen epithelial and stromal staining were analyzed semi-quantitatively.
RESULTS: Follistatin and FLRG were expressed both in normal tissue and in all the breast diseases investigated. Follistatin staining was detected in the epithelial cytoplasm and nucleus in normal, benign and malignant breast tissue, with a stronger staining intensity in the peri-alveolar stromal cells of FIB at both mRNA and protein levels. Conversely, FLRG area and intensity of mRNA and protein staining were higher both in the cytoplasm and in the nucleus of IDC epithelial cells when compared to NB, while no significant changes in the stromal intensity were observed in all the proliferative diseases analyzed.
CONCLUSION: The present findings suggest a role for follistatin in breast benign disease, particularly in FIB, where its expression was increased in stromal cells. The up regulation of FLRG in IDC suggests a role for this protein in the progression of breast malignancy. As activin displays an anti-proliferative effect in human mammary cells, the present findings indicate that an increased FST and FLRG expression in breast proliferative diseases might counteract the anti-proliferative effects of activin in human breast cancer.

Activin, a member of the transforming growth factor beta (TGFbeta) superfamily, regulates diverse processes, such as cellular growth and differentiation. There is increasing evidence that TGFbeta and its signaling effectors are key determinants of tumor cell behavior. Loss of sensitivity to TGFbeta-induced growth arrest is an important step toward malignancy. We previously characterized FLRG as an extracellular antagonist of activin. Here, we show that activin-induced growth inhibition is altered in FLRG-expressing breast cancer lines. Silencing FLRG induced growth inhibition, which is reversible upon addition of exogenous FLRG. We showed that FLRG silencing effects resulted from restoration of endogenous activin functions as shown by increased levels of phosphorylated smad2 and up-regulation of activin target gene transcripts. Furthermore, the growth inhibition induced by FLRG silencing was reversible by treatment with a soluble form of type II activin receptor. Finally, a strong expression of FLRG was observed in invasive breast carcinomas in contrast with the normal luminal epithelial cells in which FLRG was not detected. Our data provide strong evidence that endogenous FLRG contributes to tumor cell proliferation through antagonizing endogenous activin effects.

Activins are members of the transforming growth factor-beta (TGF-beta) superfamily that control many physiological processes such as cell proliferation and differentiation, immune responses, wound repair and various endocrine activities. Activins elicit these diverse biological responses by signaling via type I and type II receptor serine kinases. Recent studies have revealed details of the roles of inhibin, betaglycan, follistatin and its related protein follistatin-related gene (FLRG), Cripto and BAMBI in antagonizing activin action, and exogenous antagonists against the activin type I (SB-431542 and SB-505124) and type II (activin-M108A) receptors have been developed. Understanding how activin signaling is controlled extracellularly is the first step in providing treatment for wound healing and for disorders such as cachexia and cancer, which result from a deregulated activin pathway.

OBJECTIVE: Activin A is a multifunctional growth and cell differentiation factor produced by normal endometrium, and secreted in high amounts by endometrial adenocarcinoma. In the present study we evaluated the expression of two inhibitory activin A ligands, follistatin and follistatin-related gene (FLRG), in endometrial adenocarcinoma and in age-matched healthy human endometrium.
DESIGN AND METHODS: Atropic menopausal (n=13) and tumoral (n=9 adenocarcinoma) tissues were processed to evaluate mRNA expression levels (by semiquantitative RT-PCR) and peptide localization (by immunohistochemistry). Differences were evaluated by the unpaired t-test and assumed to be statistically significant when P<0.05.
RESULTS: Both control and tumoral endometrial samples express and localize follistatin and FLRG. However, whereas follistatin mRNA expression did not differ significantly, FLRG was significantly lower in endometrial adenocarcinoma than in healthy endometrial specimens (P<0.0001). With respect to the localization of proteins, follistatin was immunolocalized in endometrial epithelial and vascular cells both in tumoral and healthy endometrium without any significant difference in intensity. Nuclear and cytoplasmic FLRG immunolocalization was seen in glands, and only nuclear immunolocalization was found in stroma and vessels of healthy endometrium. FLRG was weakly immunostained in endometrial adenocarcinoma.
CONCLUSIONS: Whilst follistatin expression is unchanged, FLRG is down-regulated in endometrial carcinoma. As activin A is a differentiation factor of human endometrium, the present findings support an imbalance between increased activin A and decreased FLRG expression in endometrial cancer, so that the failure of the activin A pathway through FLRG may be pivotal in endometrial tumorigenesis.

Follistatin-related protein (FSRP) is a new addition to the expanding follistatin (FS)-related gene family whose members contain at least one conserved 10-cysteine follistatin domain. In contrast to other members of this family, FSRP and follistatin also share a common exon/intron domain structure, substantial primary sequence homology, and an ability to irreversibly bind activin. In this study, we further explored the hypothesis that FSRP is a functional as well as structural homologue of FS. N-terminal sequencing of recombinant FSRP revealed that signal peptide cleavage occurs within exon 1, a significant structural difference from FS, in which cleavage occurs at the exon/intron boundary. Solid-phase radioligand competition assays revealed both FS and FSRP to preferentially bind activin with the next closest TGF-beta superfamily member, bone-morphogenic protein-7, being at least 500-fold less potent. Consistent with their similar activin-binding affinities, FSRP and FS both prevented exogenous (endocrine or paracrine) activin from accessing its receptor and inducing gene transcription in bioassays. However, FS was at least 100-fold more potent than FSRP in inhibiting gene transcription and FSH release mediated by endogenously produced (autocrine) activin-A or activin-B in multiple cell systems. Finally, FSRP lacks the heparin-binding sequence found in FS, and we found that it was also unable to bind cell surface heparin sulfated proteoglycans. These findings suggest that structural differences between FSRP and FS may underlie their different neutralizating capabilities with respect to exogenous vs. endogenous activin. Taken together with our previous studies showing that activin binding is essential for FS's biological activity, the differential activities of FSRP and FS further indicate that activin binding is necessary but not sufficient to account for all of FS's actions.

We report here the molecular study of a t(11;19)(q13;p13) translocation observed in a case of B-cell chronic lymphocytic leukemia. This translocation leads to the juxtaposition of the CCND1 gene on chromosome 11 to a new transcriptional unit on chromosome 19. The cDNA of this new evolutionarily conserved gene (named FLRG for Follistatin-Related Gene) codes for a secreted glycoprotein of the follistatin-module-protein family. FLRG is expressed in a wide range of human and murine adult tissues and its expression seems to be tightly regulated during murine embryogenesis. Its transcripts could not be detected in hematopoietic cells from all lineages and in particular in cells from lymphoid B and T lineage except in the t(11;19)-carrying leukemia described here. A great variability of expression is observed among the other tumoral cell lines analysed. Besides the t(11;19)-carrying leukemia described in this work, structural rearrangements of the FLRG locus have been found in a non-Hodgkin lymphoma, suggesting that it may play a role in leukemogenesis.