Targeting the activin receptor 1C on CD4+ T cells for cancer immunotherapy.

Activins, members of the TGF-beta superfamily, have been isolated and identified in the endocrine system, but have not been substantially investigated in the context of the immune system and endocrine-unrelated cancers. Here, we demonstrated that tumor-bearing mice had elevated systemic activin levels, which correlated directly with tumor burden. Likewise, cancer patients have elevated plasma activin levels compared to healthy controls. We observed that both tumor and immune cells could be sources of activins. Importantly, our in vitro studies suggest that activins promote differentiation of naïve CD4+ cells into Foxp3-expressing induced regulatory T cells (Tregs), particularly when TGF-beta was limited in the culture medium. Database and qRT-PCR analysis of sorted major immune cell subsets in mice revealed that activin receptor 1c (ActRIC) was uniquely expressed on Tregs and that both ActRIC and ActRIIB (activin receptor 2b) were highly upregulated during iTreg differentiation. ActRIC-deficient naïve CD4+ cells were found to be defective in iTreg generation both in vitro and in vivo. Treg suppression assays were also performed, and ActRIC deficiency did not change the function or stability of iTregs. Mice lacking ActRIC or mice treated with monoclonal anti-ActRIC antibody were more resistant to tumor progression than wild-type controls. This phenotype was correlated with reduced expression of Foxp3 in CD4+ cells in the tumor microenvironment. In light of the information presented above, blocking activin-ActRIC signaling is a promising and disease-specific strategy to impede the accumulation of immunosuppressive iTregs in cancer. Therefore, it is a potential candidate for cancer immunotherapy.

The Photoperiod Regulates Granulosa Cell Apoptosis through the FSH-Nodal/ALK7 Signaling Pathway in Phodopus sungorus.

The photoperiod regulates the seasonal reproduction of mammals by affecting the follicle development, for which the granulosa cells provide nutrition. However, the underlying mechanism remains unclear. Here, Djungarian hamsters (Phodopus sungorus) were raised under different photoperiods to study the ovarian status and explore the potential mechanism of the follicle development mediated by the FSH-Nodal/ALK7 signaling pathway. Compared with the moderate daylight (MD) group, the short daylight (SD) group exhibited a significant decrease in the ovarian weight and increase in the atretic follicle number and granulosa cell apoptosis, whereas the long daylight (LD) group showed an increase in the ovarian weight, the growing follicle number, and the antral follicle number, but a decrease in the granulosa cell apoptosis. Based on these findings, the key genes of the Nodal/ALK7 signaling pathway controlling the granulosa cell apoptosis were studied using the quantitative real-time polymerase chain reaction and western blotting. In the SD group, the follicle-stimulating hormone (FSH) concentration significantly decreased and the Nodal/ALK7/Smad signaling pathways were activated, while the phosphatidylinositol 3-kinase (PIK3)/Akt signaling pathway was inhibited. The BAX expression was significantly increased, while the Bcl-xL expression was significantly decreased, leading to an increase in the caspase-3 activity, the granulosa cell apoptosis, and ovarian degeneration. However, in the LD group, the FSH concentration significantly increased, the Nodal/ALK7/Smad signaling pathway was inhibited, and the PIK3/Akt signaling pathway was activated. Taken together, our results indicate that the photoperiod can regulate the apoptosis of the granulosa cells by regulating the concentration of FSH, activating or inhibiting the Nodal/ALK7 signaling pathway, thereby affecting the ovarian function. Our research provides an important theoretical basis for understanding the photoperiod-regulated mechanisms of the mammalian seasonal reproduction.

ALK7 Inhibition Protects Osteoblast Cells Against High Glucoseinduced ROS Production via Nrf2/HO-1 Signaling Pathway.

BACKGROUND: Some studies demonstrated that under high-glucose (HG) condition, osteoblasts develop oxidative stress, which will impair their normal functions. The effects of activin receptor-like kinase 7 (ALK7) silencing on HG-induced osteoblasts remained unclear. OBJECTIVE: The aim of this study was to explore the effect of ALK7 on HG-induced osteoblasts. METHODS: MC3T3-E1 cells were treated with different concentrations of HG (0, 50, 100, 200 and 300mg/dL), and the cell viability was detected using cell counting kit-8 (CCK-8). HG-treated MC3T3-E1 cells were transfected with siALK7 or ALK7 overexpression plasmid or siNrf2, and then the viability and apoptosis were detected by CCK-8 and flow cytometry. The levels of Reactive Oxygen Species (ROS), collagen I and calcification nodule were determined by oxidative stress kits, Enzyme-linked immunosorbent assay and Alizarin red staining. The expressions of NF-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and osteoblast-associated genes were determined by quantitative real-time PCR (qRT-PCR) and Western blot. RESULTS: Cell viability was reduced with HG treatment. Silencing ALK7 inhibited the effect of HG on increasing cell apoptosis and ROS production, reduced cell viability, mineralized nodules, and downregulated collagen I and osteoblast-associated genes expression in MC3T3-E1 cells. ALK7 silencing activated the Nrf2/HO-1 signaling pathway by affecting expressions of HO-1 and Nrf2. ALK7 overexpression had the opposite effects. In addition, siNrf2 partially reversed the effects of ALK7 silencing on HG-induced MC3T3-E1 cells. CONCLUSION: ALK7 silencing protected osteoblasts under HG condition possibly through activating the Nrf2/HO-1 pathway.

Epstein-Barr virus microRNA BART10-3p promotes dedifferentiation and proliferation of nasopharyngeal carcinoma by targeting ALK7.

Non-keratinizing nasopharyngeal carcinoma, the major subtype of nasopharyngeal carcinoma, is characterized by low differentiation and a close relation to Epstein-Barr virus infection, which indicates a link between Epstein-Barr virus oncogenesis and loss of differentiation, and raises our interest in investigating the involvement of Epstein-Barr virus in nasopharyngeal carcinoma dedifferentiation. Our previous study showed abundant expression of an Epstein-Barr virus-encoded microRNA, BART10-3p, in nasopharyngeal carcinoma tissues, but the association between BART10-3p and nasopharyngeal carcinoma differentiation remains unknown. Here, we examined the expression and prognostic value of BART10-3p, and undertook bioinformatics analysis and functional assays to investigate the influence of BART10-3p on nasopharyngeal carcinoma differentiation and proliferation and the underpinning mechanism. Microarray analysis identified BART10-3p as the most significantly upregulated Epstein-Barr virus-encoded microRNA in nasopharyngeal carcinoma tissues and the upregulation was confirmed in two public datasets. The expression of BART10-3p was an independent unfavorable prognosticator in nasopharyngeal carcinoma and its integration with the clinical stage showed improved prognosis predictive performance. Bioinformatics analysis suggested a potential role of BART10-3p in tumor differentiation and progression. Functional assays demonstrated that BART10-3p could promote nasopharyngeal carcinoma cell dedifferentiation, epithelial-mesenchymal transition, and proliferation in vitro, and tumorigenicity in vivo. Mechanistically, BART10-3p directly targeted the 3'UTR of ALK7 and suppressed its expression. Reconstitution of ALK7 rescued BART10-3p-induced malignant phenotypes. Overall, our study demonstrates that BART10-3p promotes dedifferentiation and proliferation of nasopharyngeal carcinoma by targeting ALK7, suggesting a promising therapeutic opportunity to reverse the malignant phenotypes of nasopharyngeal carcinoma.

ALK7 Acts as a Positive Regulator of Macrophage Activation through Down-Regulation of PPARγ Expression.

AIM: Activin receptor-like kinase 7 (ALK7) acts as a key receptor for TGF-ß family members, which play important roles in regulating cardiovascular activity. However, ALK7's potential role, and underlying mechanism, in the macrophage activation involved in atherogenesis remain unexplored. METHODS: ALK7 expression in macrophages was tested by RT-PCR, western blot, and immunofluorescence co-staining. The loss-of-function strategy using AdshALK7 was performed for functional study. Oil Red O staining was used to observe the foam cell formation, while inflammatory mediators and genes related to cholesterol efflux and influx were determined by RT-PCR and western blot. A PPARγ inhibitor (G3335) was used to reveal whether PPARγ was required for ALK7 to affect macrophage activation. RESULTS: The results exhibited upregulated ALK7 expression in oxidized low-density lipoprotein (Ox-LDL) induced bone marrow derived macrophages (BMDMs) and mouse peritoneal macrophages (MPMs), isolated from ApoE-deficient mice, while ALK7's strong immunoreactivity in BMDMs was observed. ALK7 knockdown significantly attenuated pro-inflammatory, but promoted anti-inflammatory, macrophage markers expression. Additionally, ALK7 silencing decreased foam cell formation, accompanied by the up-regulation of ABCA1 and ABCG1 involved in cholesterol efflux but the down-regulation of CD36 and SR-A implicated in cholesterol influx. Mechanistically, ALK7 knockdown upregulated PPARγ expression, which was required for the ameliorated effect of ALK7 silencing macrophage activation. CONCLUSIONS: Our study demonstrated that ALK7 was a positive regulator for macrophage activation, partially through down-regulation of PPARγ expression, which suggested that neutralizing ALK7 might be promising therapeutic strategy for treating atherosclerosis.

A set of microRNAs coordinately controls tumorigenesis, invasion, and metastasis.

MicroRNA-mediated gene regulation has been implicated in various diseases, including cancer. This study examined the role of microRNAs (miRNAs) during tumorigenesis and malignant progression of pancreatic neuroendocrine tumors (PanNETs) in a genetically engineered mouse model. Previously, a set of miRNAs was observed to be specifically up-regulated in a highly invasive and metastatic subtype of mouse and human PanNET. Using functional assays, we now implicate different miRNAs in distinct phenotypes: miR-137 stimulates tumor growth and local invasion, whereas the miR-23b cluster enables metastasis. An algorithm, Bio-miRTa, has been developed to facilitate the identification of biologically relevant miRNA target genes and applied to these miRNAs. We show that a top-ranked miR-137 candidate gene, Sorl1, has a tumor suppressor function in primary PanNETs. Among the top targets for the miR-23b cluster, Acvr1c/ALK7 has recently been described to be a metastasis suppressor, and we establish herein that it is down-regulated by the miR-23b cluster, which is crucial for its prometastatic activity. Two other miR-23b targets, Robo2 and P2ry1, also have demonstrable antimetastatic effects. Finally, we have used the Bio-miRTa algorithm in reverse to identify candidate miRNAs that might regulate activin B, the principal ligand for ALK7, identifying thereby a third family of miRNAs-miRNA-130/301-that is congruently up-regulated concomitant with down-regulation of activin B during tumorigenesis, suggestive of functional involvement in evasion of the proapoptotic barrier. Thus, dynamic up-regulation of miRNAs during multistep tumorigenesis and malignant progression serves to down-regulate distinctive suppressor mechanisms of tumor growth, invasion, and metastasis.

ALK7 Signaling Manifests a Homeostatic Tissue Barrier That Is Abrogated during Tumorigenesis and Metastasis.

Herein, we report that the TGFß superfamily receptor ALK7 is a suppressor of tumorigenesis and metastasis, as revealed by functional studies in mouse models of pancreatic neuroendocrine and luminal breast cancer, complemented by experimental metastasis assays. Activation in neoplastic cells of the ALK7 signaling pathway by its principal ligand activin B induces apoptosis. During tumorigenesis, cancer cells use two different approaches to evade this barrier, either downregulating activin B and/or downregulating ALK7. Suppressing ALK7 expression additionally contributes to the capability for metastatic seeding. ALK7 is associated with shorter relapse-free survival of various human cancers and distant-metastasis-free survival of breast cancer patients. This study introduces mechanistic insights into primary and metastatic tumor development, in the form of a protective barrier that triggers apoptosis in cells that are not "authorized" to proliferate within a particular tissue, by virtue of those cells expressing ALK7 in a tissue microenvironment bathed in its ligand.

MicroRNA-454 contributes to sustaining the proliferation and invasion of trophoblast cells through inhibiting Nodal/ALK7 signaling in pre-eclampsia.

MicroRNAs (miRNAs) are emerging as important regulators in the pathogenesis of pre-eclampsia (PE). Recent evidence has reported that miR-454 plays an important role in regulating cell proliferation and invasion. The decreased proliferation and invasion of trophoblast cells contribute to the pathogenesis of PE. However, whether miR-454 is involved in the regulation of trophoblast cell proliferation and invasion remains unknown. In this study, we aimed to investigate the potential role and underlying mechanism of miR-454 in regulating trophoblast cell proliferation and invasion in vitro. We found that miR-454 expression was significantly decreased in placental tissues from PE patients compared to controls. Transfection of miR-454 mimics promoted the proliferation, reduced the apoptosis, and increased invasion of trophoblast cells, while transfection of miR-454 inhibitor showed opposite effects. Bioinformatics analysis showed that activin receptor-like kinase 7 (ALK7) was a potential target gene of miR-454. Dual-luciferase reporter assay showed miR-454 directly targeted the 3'-untranslated region of AKL7. Further experiments showed that miR-454 negatively regulated ALK7 expression. Interestingly, transfection of miR-454 mimics significantly abrogated the inhibitory effect of Nodal on trophoblast cell proliferation and invasion. Moreover, overexpression of ALK7 markedly reversed the promotion effect of miR-454 on trophoblast cell proliferation and invasion. Overall, our results suggest that miR-454 promotes the proliferation and invasion of trophoblast cells by downregulation of ALK7. Our study suggests that miR-454 may play critical roles in the pathogenesis of PE and serve as a potential therapeutic target for treatment of PE.

ALK7 expression in prolactinoma is associated with reduced prolactin and increased proliferation.

Prolactinoma represents the most frequent hormone-secreting pituitary tumours. These tumours appear in a benign form, but some of them can reach an invasive and aggressive stage through an unknown mechanism. Discovering markers to identify prolactinoma proliferative and invading character is therefore crucial to develop new diagnostic/prognostic strategies. Interestingly, members of the TGFß-Activin/BMP signalling pathways have emerged as important actors of pituitary development and adult function, but their role in prolactinomas remains to be precisely determined. Here, using a heterotopic allograft model derived from a rat prolactinoma, we report that the Activins orphan type I receptor ALK7 is ectopically expressed in prolactinomas-cells. Through immunohistological approaches, we further confirm that normal prolactin-producing cells lack ALK7-expression. Using a series of human tumour samples, we show that ALK7 expression in prolactinomas cells is evolutionary conserved between rat and human. More interestingly, our results highlight that tumours showing a robust expression of ALK7 present an increased proliferation as address by Ki67 expression and retrospective analysis of clinical data from 38 patients, presenting ALK7 as an appealing marker of prolactinoma aggressiveness. Beside this observation, our work pinpoints that the expression of prolactin is highly heterogeneous in prolactinoma cells. We further confirm the contribution of ALK7 in these observations and the existence of highly immunoreactive prolactin cells lacking ALK7 expression. Taken together, our observations suggest that Activin signalling mediated through ALK7 could therefore contribute to the hormonal heterogeneity and increased proliferation of prolactinomas.

Overexpression of Activin Receptor-like Kinase 7 in Breast Cancer Cells Is Associated with Decreased Cell Growth and Adhesion.

AIM: To examine the expression and function of activin receptor-like kinase 7 (ALK7) in breast cancer, its association with disease prognosis, and its impact on breast cancer cell function. PATIENTS AND METHODS: A cohort of patients with breast cancer were examined for ALK7 expression in association with pathological and clinical aspects. In vitro cell assays of ALK7 were investigated using an expression plasmid. RESULTS: Overall higher levels of ALK7 transcripts were seen in the breast cancer samples vs. normal tissue. However, within the cancer cohort, lower levels of ALK7 transcript were associated with poor prognosis. Patients with lower expression of ALK7 also had shorter survival. Overexpression of ALK7 reduced proliferation and adhesion of breast cancer cells in vitro. We found that overexpressed ALK7 had complex effects on the MCF-7 cell sensitivity to chemotherapy drugs. CONCLUSION: Decreased expression of ALK7 in breast cancer is correlated with poor prognosis. ALK7 is a negative regulator of adhesion and proliferation of breast cancer cells. This suggests that ALK7 is a potential tumor suppressor in breast cancer.

Overexpression of B2M and loss of ALK7 expression are associated with invasion, metastasis, and poor-prognosis of the pancreatic ductal adenocarcinoma.

BACKGROUND: Approximately 80% of patients with pancreatic ductal adenocarcinoma (PDAC) have metastatic disease with poor prognosis, but clinically available markers have not yet been identified. OBJECTIVE: In this study, we investigated the expression of B2M and ALK7 in 106 PDACs compared to precursor lesions of the pancreas. METHODS: Immunohistochemistry was used to detect B2M and ALK7 protein expression. RESULTS: Positive B2M expression was significantly higher, while positive expression of ALK7 was significantly lower in PDAC than in precursor lesions (p < 0.01 or p < 0.001). Positive B2M expression was also significantly higher, while positive ALK7 expression was significantly lower in cases with well-differentiated adenocarcinoma, small tumor mass, no-metastasis of the lymph node, no-invasion of regional tissues, and TNM I or II stage disease than in cases having poorly-differentiated adenocarcinoma, large tumor mass, with metastasis and invasion, and TNM stage III or IV stage disease (p < 0.01). Univariate Kaplan-Meier analysis showed that B2M overexpression (p < 0.001), but lack of ALK7 expression (p < 0.001) was significantly associated with shorter overall survival. Cox multivariate analysis showed that differentiation, tumor mass, lymph node metastasis, invasion, TNM stage, and B2M levels negatively correlated with overall survival. In contrast, ALK7 level positively correlated with overall survival. Positive B2M and negative ALK7 expression are poor prognostic factors in PDAC patients. CONCLUSIONS: B2M and ALK7 might be important biological markers involved in the carcinogenesis, metastasis, invasion, and prognosis of PDAC.

ALK7 protects against pathological cardiac hypertrophy in mice.

AIMS: Activin receptor-like kinase 7 (ALK7), one of the type I transforming growth factor-ß receptors, is expressed in various tissues, including the heart. However, the participation of ALK7 in the regulation of cardiac hypertrophy has not yet been studied. Here, we sought to determine the regulatory role and underlying mechanisms of ALK7 in cardiac hypertrophy. METHODS AND RESULTS: We performed aortic banding (AB) in ALK7-knockout mice, cardiac-specific ALK7-transgenic mice, and the wild-type littermates of these mice. Cardiac hypertrophy was evaluated using pathological analysis, echocardiographic measurement, haemodynamic measurement, and molecular analysis. Our results revealed that ALK7 disruption led to an aggravated cardiac hypertrophic response that was accompanied by increased cardiac fibrosis and reduced contractile function, whereas cardiac-specific ALK7 overexpression exhibited the opposite phenotype in response to pressure overload. Similarly, ALK7 protected against angiotensin II-induced cardiomyocyte hypertrophy in vitro. Mechanistically, we demonstrated that ALK7-dependent cardioprotection was mediated largely through inhibition of the MEK-ERK1/2 signalling pathway. CONCLUSION: Our data suggest that ALK7 acts as a novel regulator of pathological cardiac hypertrophy via the negative regulation of MEK-ERK1/2 signalling and may serve as a potential therapeutic target for pathological cardiac hypertrophy.

A novel crosstalk between Alk7 and cGMP signaling differentially regulates brown adipocyte function.

OBJECTIVE: Obesity is an enormous burden for patients and health systems world-wide. Brown adipose tissue dissipates energy in response to cold and has been shown to be metabolically active in human adults. The type I transforming growth factor ß (TGFß) receptor Activin receptor-like kinase 7 (Alk7) is highly expressed in adipose tissues and is down-regulated in obese patients. Here, we studied the function of Alk7 in brown adipocytes. METHODS: Using pharmacological and genetic tools, Alk7 signaling pathway and its effects were studied in murine brown adipocytes. Brown adipocyte differentiation and activation was analyzed. RESULTS: Alk7 is highly upregulated during differentiation of brown adipocytes. Interestingly, Alk7 expression is increased by cGMP/protein kinase G (PKG) signaling, which enhances brown adipocyte differentiation. Activin AB effectively activates Alk7 and SMAD3 signaling. Activation of Alk7 in brown preadipocytes suppresses the master adipogenic transcription factor PPARγ and differentiation. Stimulation of Alk7 during late differentiation of brown adipocytes reduces lipid content and adipogenic marker expression but enhances UCP1 expression. CONCLUSIONS: We found a so far unknown crosstalk between cGMP and Alk7 signaling pathways. Tight regulation of Alk7 is required for efficient differentiation of brown adipocytes. Alk7 has differential effects on adipogenic differentiation and the development of the thermogenic program in brown adipocytes.

Nodal promotes glioblastoma cell growth.

Nodal is a member of the transforming growth factor-ß (TGF-ß) superfamily that plays critical roles during embryogenesis. Recent studies in ovarian, breast, prostate, and skin cancer cells suggest that Nodal also regulates cell proliferation, apoptosis, and invasion in cancer cells. However, it appears to exert both tumor-suppressing and tumor-promoting effects, depending on the cell type. To further understand the role of Nodal in tumorigenesis, we examined the effect of Nodal in glioblastoma cell growth and spheroid formation using U87 cell line. Treatment of U87 with recombinant Nodal significantly increased U87 cell growth. In U87 cells stably transfected with the plasmid encoding Nodal, Smad2 phosphorylation was strongly induced and cell growth was significantly enhanced. Overexpression of Nodal also resulted in tight spheroid formation. On the other hand, the cells stably transfected with Nodal siRNA formed loose spheroids. Nodal is known to signal through activin receptor-like kinase 4 (ALK4) and ALK7 and the Smad2/3 pathway. To determine which receptor and Smad mediate the growth promoting effect of Nodal, we transfected siRNAs targeting ALK4, ALK7, Smad2, or Smad3 into Nodal-overexpressing cells and observed that cell growth was significantly inhibited by ALK4, ALK7, and Smad3 siRNAs. Taken together, these findings suggest that Nodal may have tumor-promoting effects on glioblastoma cells and these effects are mediated by ALK4, ALK7, and Smad3.