The activin type II receptor (ACVR2) gene is a putative tumor suppressor gene that is frequently mutated in microsatellite-unstable colon cancers (MSI-H colon cancers). ACVR2 is a member of the transforming growth factor (TGF)-β type II receptor (TGFBR2) family and controls cell growth and differentiation. SMAD proteins are major intracellular effectors shared by ACVR2 and TGFBR2 signaling; however, additional shared effector mechanisms remain to be explored. To discover novel mechanisms transmitting the ACVR2 signal, we restored ACVR2 function by transfecting wild-type ACVR2 (wt-ACVR2) into a MSI-H colon cancer cell line carrying an ACVR2 frameshift mutation. The effect of ACVR2 restoration on cell growth, SMAD phosphorylation, and global molecular phenotype was then evaluated. Decreased cell growth was observed in wt-ACVR2 transfectants relative to ACVR2-deficient vector-transfected controls. Western blotting revealed higher expression of phosphorylated SMAD2 in wt-ACVR2 transfectants versus controls, suggesting cells deficient in ACVR2 had impaired SMAD signaling. Microarray-based differential expression analysis revealed substantial ACVR2-induced overexpression of genes implicated in the control of cell growth and tumorigenesis, including the activator protein (AP)-1 complex genes JUND, JUN, and FOSB, as well as the small GTPase signal transduction family members, RHOB, ARHE, and ARHGDIA. Overexpression of these genes is shared with TGFBR2 activation. This observed similarity between the activin and TGF-β signaling systems suggests that activin may serve as an alternative activator of TGF-β effectors, including SMADs, and that frameshift mutation of ACVR2 may contribute to MSI-H colon tumorigenesis via disruption of alternate TGF-β effector pathways.

The activin type II receptor (ACVR2) gene encodes the type II subunit of the activin receptor complex. The type II subunit is essential to activin-mediated signaling; the extracellular binding domain binds to activin, and the intracellular kinase domain activates the type I subunit that activates SMADs (reviewed in ref. 1). Our previous studies detected very frequent frameshift mutations in the A8 tract of exon 10 of ACVR2 in gastrointestinal cancers with frequent microsatellite instability (MSI-H; colon cancers, 58%; gastric cancers, 44%; ref. 2). Another study identified biallelic mutation of ACVR2 in 86% of MSI-H colon and pancreatic cancer xenografts and cell lines (3). Loss of ACVR2 protein was also reported in the majority of MSI-H tumors harboring frameshift mutation at the polyadenine tract of exon 10 of ACVR2(4).

Activin signaling is involved in the regulation of apoptosis, differentiation, proliferation, and cell migration in many tissues, including epithelium, lymphocytes, prostate cancer, breast, vascular endothelium, and liver (reviewed in ref. 1). The activin ligand binds to a heterodimeric transmembrane activin-receptor complex with serine/threonine kinase activity that consists of type I and type II subunits (reviewed in ref. 1). This receptor complex belongs to the transforming growth factor (TGF)-β receptor family and, as does the TGF-β receptor complex, takes the SMAD family of proteins as its downstream signal transducers (5). On binding to activin, the activin-receptor complex phosphorylates SMAD2 and SMAD3 in the cytoplasm, resulting in their activation. Phosphorylated SMADs form a complex with SMAD4 and activate transcription of downstream genes (6).

Some members of the activin signaling pathway have been implicated as tumor suppressor genes. SMAD4 has been reported as a tumor suppressor in human pancreatic and colon cancers (7). SMAD2 is mutated in colon and lung cancers (5). Similarly, mutational inactivation of the activin type I receptor gene (ACVR1) have been observed in pancreatic cancers (8). SMAD3 null mice develop metastatic colon cancers (9). A dominant negative mutant ACVR2 also abolishes activin-mediated erythroid differentiation (10). Finally, a recent study showed that activin signaling exerts growth-suppressive effects in colon cancer cells (11).

Microsatellite instability occurring within coding regions underlies tumorigenesis in cancers with frequent microsatellite instability (MSI-H cancers; ref. 12). Frequent frameshift mutations in MSI-H cancers have been reported in TGFBR2 as well as ACVR2(2, 13). To characterize the influence of ACVR2 gene frameshift mutation on MSI-H colon cancer cells, we analyzed changes in global molecular phenotype after re-expression of constitutively active wild-type (wt)-ACVR2 using the following: (a) microarray-based gene expression profiling; and (b) analysis of phospho-SMAD2 (p-SMAD2) expression.

Plasmid Constructs.

The entire coding region of wt-ACVR2 was PCR-amplified and cloned into the plasmid vector pEF6/V5-His-TOPO (Invitrogen, Carlsbad, CA). The plasmid construct was purified with the HiSpeed Plasmid Midi Kit (Qiagen, Valencia, CA) and analyzed by sequencing and restriction enzyme digestion to confirm the sequence and direction of the insert.

Transfections.

The MSI-H colon cancer cell line HCT-15 was used for transfection experiments. HCT-15 carries a biallelic ACVR2 frameshift mutation at an A8 tract in exon 10 and is inactivated in TGFBR2 by mutations in both alleles (deletion at an A10 tract in exon 3 and a missense mutation in exon 5: L452P; ref. 14). HCT-15 were cultured in growth medium at 37°C with 5% CO2. Cells were reseeded in six-well plates at a density of 2.5 × 105 cells per well one day before transfection. Lipofectamine 2000 (Invitrogen) was used to transfect 4 μg of the expression plasmid encoding wt-ACVR2 as well as control plasmid containing the LacZ gene (pEF6/V5-His-TOPO/lacZ). Ten stable wt-ACVR2 and eight stable vector-control transfectants were selected by expanding single cells in growth media containing 14 μg/mL Blasticidin S HCl (Invitrogen).

Cell Growth Assay.

Direct cell counting was done as follows: Three wt-ACVR2-transfected clones and three control vector-transfected clones were plated at a density of 5 × 103 cells per well in 96-well plates in growth media in triplicates at day 0 and counted every day over a 5-day period.

Bromodeoxyuridine (BrdUrd) incorporation assay was done as follows: The experiment was done with Cell Proliferation ELISA BrdUrd (colorimetric) Kit (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s protocol. Detailed protocol is available in the Supplementary Methods.

Antibodies.

The rabbit polyclonal antibody against ACVR2 has been produced by Washington Biotechnology (Baltimore, MD). Briefly, two rabbits were twice immunized with a synthetic peptide (nwekdrtnqtgvepcy; 36–51) corresponding to the extracellular domain of ACVR2. Six weeks later, the antibodies were affinity chromatography purified from the antisera of the rabbit. Rabbit polyclonal antibodies against SMAD2/3 and p-SMAD2 were purchased from Upstate Biotech (Lake Placid, NY) and Cell Signaling Technology (Beverly, MA; used for activin stimulation experiments). Rabbit polyclonal antiactin antibodies were purchased from Santa Cruz Biotechnology Biotech (Santa Cruz, CA).

Western Blotting.

Cell lysates were pelleted and then resuspended in 200 μL cell lysis buffer [NaCl 149 mmol/L, NP40 0.01%, Tris 50 mmol/L (pH 7.8), and protease inhibitor cocktail 0.5% (Sigma, St. Louis, MO)]. The protein concentration was determined with the BCA Protein Assay Kit (Pierce, Rockford, IL) with human serum albumin as a standard. The samples were electrophoresed in 10% NuPAGE gel (Invitrogen) and transferred onto polyvinylidene difluoride membrane (Invitrogen). The membranes were immunoblotted with anti-ACVR2 polyclonal antibody (1:5,000 dilution), anti-Smad2/3 antibody (2 μg/mL), and antiphosphorylated Smad2 (1:1,500 dilution). Target protein bands were visualized with ECL Western Blotting detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). The antiactin antibodies were used as a loading control.

For activin stimulation experiments, wt-ACVR2- and control vector transfectants were plated in six-well plates at a density of 106 cells per well and cultured for 24 hours. The cells were then starved overnight in serum-free medium before the stimulation with 10 or 100 ng of recombinant activin A (Calbiochem, San Diego, CA). The protein extracts from both untreated and treated cells were obtained after 1 or 2 hours of stimulation with PhophoSafe Extraction Buffer (Novagen, San Diego, CA). HeLa cells treated with 100 ng of TGF-β1 (Roche Applied Science) were used as a positive control. Thirty 30 micrograms of total protein extracts were electrophoresed and transferred as described above. The membranes were immunoblotted with anti-phospho–SMAD2 (1 μg/mL) or anti-SMAD2/3 (1 μg/mL) antibody.

Real-time Quantitative Reverse Transcription (RT)-PCR.

Total RNA was extracted with TRIzol reagents (Invitrogen) and was treated with RNase-free DNase on the RNeasy columns (Qiagen).

ACVR2 expression was measured with TaqMan method-based real-time quantitative RT-PCR as described in the Supplementary Methods. β-actin was used as a normalization control. The cDNA from untransfected HCT-15 cells was used as a quantification standard. The formula for normalization was as follows: ratio of sample to reference cDNA = [ACVR2(s)/ACVR2(r)]/[(β-actin(s)/(β-actin(r)], where ACVR2(s) and ACVR2(r) were expression levels of ACVR2 in the samples and reference cDNA, respectively, and β-actin(s) and β-actin(r) were β-actin RNA expression levels in the samples and reference. For validation of microarray results, real-time quantitative one-step RT-PCR analysis was done with Quantitect SYBR Green RT-PCR kit (Qiagen) on iCycler (Bio-Rad, Hercules, CA). Normalization to β-actin expression level was done as well.

The sequences of all of the primers and probes are shown in the Supplementary Table 2. Detailed real-time quantitative PCR methods are available in the Supplementary Methods.

Microarray Preparation and Hybridization.

Microarray analysis of three wt-ACVR2-transfected and three control vector-transfected clones was done as described previously (15). Briefly, amplified RNA was obtained from 20 to 50 μg of total RNA from each clone with a T7-based protocol and was labeled with Cy5 with random primers and reverse transcriptase. The reference sample was a pool of amplified RNAs from eight human malignant cell lines labeled with Cy3. Each Cy5-labeled specimen probe and the Cy3-labeled reference probe were cohybridized to a microarray slide containing 8,064 sequence-verified human cDNA clones. Probe preparation and hybridization was done individually for each sample clones. After hybridization, each slide was scanned with a GenePix 4000A dual-color slide scanning system (Axon Instruments, Union City, CA).

Data Analysis.

We performed both within-slide and between-slide normalization before analysis with LOWESS curve-fitting methods (15). Significance analysis of microarrays was applied to the Lowess-normalized log-scaled data to select genes that were significantly differentially expressed between wt-ACVR2-transfected and control vector-transfected clones (15). Significantly differential expression was determined based on significance analysis of microarrays score, a score assigned to each gene on the basis of its fold change of average expression levels between the wt-ACVR2 transfectants and the control vector transfectants relative to the cumulative SDs of expression levels for wt-ACVR2 transfectants and control vector transfectants. In this study, only genes with a significance analysis of microarrays score higher than 2 (up-regulation in wt-ACVR2 transfectants) or smaller than −2 (down-regulation in wt-ACVR2 transfectants) were classified as significant.

Genes previously related to TGF-β or activin signaling were identified by online database searches. The following web sites were used for this search: PubMed (http://www.ncbi.nim.nih.gov), Stanford SOURCE (http://genome-www5.stanford.edu/cgi-bin/source/sourceResult), Human Genome Browser Gateway (http://genome.ucsc.edu), and GeneCards (http://bioinfo.weizmann.ac.il/cards-bin/cardsearch.pl).

We established a model system for studying wt-ACVR2 function by reconstituting its activity in HCT-15 colon cancer cells, which are ACVR2-deficient secondary to a native biallelic nonsense mutation. We then analyzed the global molecular phenotype of these cells before and after ACVR2 reconstitution. In addition, we evaluated the impact of ACVR2 mutation and restoration on its known downstream effector, SMAD2.

Confirmation of Successful ACVR2 Reconstitution.

After stable transfection and selection of 18 single-cell clonal transfectants (10 ACVR2- and eight control vector-transfected clones), ACVR2 mRNA levels were measured by real-time quantitative RT-PCR analysis. Measurements in all of the 10 wt-ACVR2 stable transfectants confirmed that ACVR2 mRNA levels were higher than in all of the eight control vector-transfected clones, as shown in Fig. 1,A. Next, ACVR2 protein expression level was evaluated by Western blotting. ACVR2 protein was detectable in all of the 10 wt-ACVR2 transfectants, and higher levels of ACVR2 protein were observed in all of the 10 wt-ACVR2 transfectants than in vector control transfectants (Fig. 1 B). On the basis of the data for ACVR2 mRNA and protein levels, the three wt-ACVR2 transfectants exhibiting the highest mRNA expression levels were selected for additional analyses.

Effect of ACVR2 on Cell Growth.

Restoration of wt-ACVR2 function in HCT-15 colon cancer cells resulted in slower cell growth measured by direct cell counting. The difference in growth between wt-ACVR2 transfectants and control vector transfectants was statistically significant for days 3, 4, and 5 (P < 0.05, Student’s t test; Fig. 2). The calculated doubling time was longer in the wt-ACVR2-transfected cells (20 hours) than in the controls (17.4 hours). Additionally, BrdUrd incorporation rate during DNA replication was significantly decreased in wt-ACVR transfectants compared with controls (P < 0.05, Student’s t test; data not shown).

Effect of ACVR2 on SMAD Signaling.

The effect of ACVR2 mutation and restoration on the known target downstream effector, SMAD2, was analyzed by Western blotting. Cell lysates from wt-ACVR2-transfected and untransfected (i.e., native mutant) HCT-15 cells were analyzed for expression of both total SMAD2 and its phosphorylated form (p-SMAD2). The p-SMAD2 expression was higher in wt-ACVR2-transfected than in untransfected HCT-15 cells, whereas total SMAD2 protein levels were identical in both (Fig. 3,A). We also evaluated the induction of SMAD2 phosphorylation by activin stimulation. A dose- and time-dependent increase in p-SMAD2 expression was observed in response to activin in wt-ACVR2 transfectants, whereas no effect of activin stimulation on p-SMAD2 level was observed in control vector transfectants (Fig. 3 B).

Effect of ACVR2 on Global Molecular Phenotype.

To additionally delineate the downstream effects of ACVR2 activation, and to provide insights into candidate downstream pathways discrete from SMAD signaling, we performed gene expression profiling using cDNA microarrays. In these experiments, three clonal wt-ACVR2 transfectants were compared with three clonal control vector transfectants. Significance analysis of microarray was used to select genes that were significantly differentially expressed between these two groups (Table 1; Supplementary Table 1). We eliminated the possibility that modification in gene expression was due to transfection reagents, selection media, or antibiotics by using control vector-transfected clones, rather than parental cells, in this gene expression comparison. ACVR2 expression in wt-ACVR2-transfected cells was the highest of all 8,064 genes on the microarrays. This result was not only expected, but also served as a validation of the cDNA microarray and model system developed in this study.

Genes significantly influenced by wt-ACVR2 transfection are shown in Table 1 (induced genes) and in Supplementary Table 1 (suppressed genes). These included genes induced by growth factors (e.g., RHOB, MAPK6, HGS, and PPAP2B); negative regulators of cell proliferation, such as BTG1, PMP22, or HGS; genes implicated in cellular growth regulation (e.g., CYR61, RHOB, GPC1, JUN, INHA, PPAP2B, and GPC1); and genes involved in intercellular adhesion (e.g., CLSTN1, LAMP2, PVRL3, PVRL2, MLLT4, ARHGDIA). A series of signal transducers, including ARHE, MAPK6, LTB, PPP2R2C, PP1R3A, MAP2K3, RAB6A, as well as several regulators of transcription (e.g., JUN, JUND, FOSB, ATF3, JUNB, EGR1, VGLL1, CEBPA, MSX1, and IRF1) were also induced by wt-ACVR2. Expression of the transcriptional repressor ATF3 was increased by wt-ACVR2. Proapoptotic genes such as NR4A1, DUSP2, and TNFRSF10C were overexpressed after wt-ACVR2 restoration, whereas the antiapoptotic gene BIRC5 was down-regulated.

To validate our microarray results, we performed real-time quantitative RT-PCR analysis. Six genes found to be up-regulated in wt-ACVR2 transfectants by cDNA microarrays (ARHE, ARHGDI, CYR61, FOSB, JUN, and JUND) were analyzed. All six of these genes exhibited increased mRNA levels in wt-ACVR2 transfectants compared with the control vector transfectants, confirming our cDNA microarray results (Supplementary Figure).

The involvement of activin signaling disruption in the origin or progression of human digestive tract cancer has been suspected based on the high ACVR2 and ACVR1 mutation rate in various tumors discovered recently (2, 3, 4, 8). However, mechanisms of activin signaling and the significance of the ACVR2 mutation in human tumorigenesis have not yet been fully elucidated. Activin and TGF-β share the same receptor binding properties, and their receptors exhibit the same substrate specificity, namely phosphorylation and activation of SMAD2 and SMAD3 (5). Because both TGF-β and activin use the same set of SMADs, it is conceivable that they share common regulatory mechanisms. Therefore, the identification of genes transcriptionally regulated by ACVR2 and elucidation of the molecular mechanisms responsible for this transcriptional regulation will bring us closer to a better understanding of both the activin and TGF-β signaling pathways. Gene expression profiling has been used by a variety of investigators to explore genetic events involving TGF-β signaling (16, 17, 18, 19). The current study used this approach to identify new participants in the activin signaling pathway and to explore their commonality with those of the TGF-β signaling pathway.

Initially, we evaluated the effect of restoration of wt-ACVR2 function in a colon cancer cell line, HCT-15, carrying a biallelic ACVR2 frameshift mutation at the mutational hotspot where the majority of human primary MSI-H digestive tract cancers demonstrate ACVR2 frameshift mutation (2, 3, 4). We showed that restoration of activin signaling by wt-ACVR2 transfection in this cell line resulted in increased SMAD2 phosphorylation in response to activin stimulation. This finding establishes that this biallelic mutation impairs signal transduction and implicates loss of ACVR2-mediated SMAD signaling in MSI-H colon cancer. Furthermore, the restoration of wt-ACVR2 function caused decreased cell growth in MSI-H colon cancer cells in vitro.

Global molecular phenotyping revealed numerous similarities between signaling via TGFBR2 and via ACVR2. For example, the AP-1 complex members including FOS and JUN have been implicated in signaling initiated by TGF-β (20). In the current study, AP-1 complex members JUN, JUND, JUNB, and FOSB were up-regulated by wt-ACVR2, suggesting that activin and TGF-β signaling share AP-1 involvement as effector mechanisms. Phosphorylated JUN and JUND have higher DNA-binding affinities than their nonphosphorylated counterparts, making them logical targets for the phosphorylation-mediated signaling shared by TGFBR2 and ACVR2 (20). Moreover, JUND is an inhibitor of normal intestinal mucosal growth in vivo and plays a critical role in the negative control of epithelial cell renewal under physiologic and pathological conditions (21). Similarly, JUN is a regulator of transcription, cell growth and maintenance, and interacts with the SMAD3/4 heterodimer (20). Another newly observed similarity between ACVR2 and TGFBR2 signaling concerns the up-regulation of genes induced or activated by growth factors. For example, the Rho protein family member RHOB is rapidly induced by TGF-β, epidermal growth factor, and platelet-derived growth factor (22). In the current study, RHOB was up-regulated in wt-ACVR2 transfected cells. RHOB is an immediate-early gene implicated in growth control as a potent inhibitor of malignant transformation as well as a suppressor of tumor growth and has been suggested to be a novel mechanism of tumor suppression by TGF(18, 19, 22). Interestingly, two other genes involved in Rho protein signaling, ARHE and ARHGDIA, were also up-regulated in wt-ACVR2 transfectants. Transcriptional repressor ATF3 is a TGF-β–inducible factor that forms a complex with SMAD3 (23). ATF3 expression was found to be increased by TGF-β but not by bone morphogenetic protein (23). In the current study, ATF3 was overexpressed in wt-ACVR2 transfectants, suggesting that activin is another TGF-β family member that can induce the expression of ATF3. Finally, in the current study, wt-ACVR2 restoration up-regulated a growth factor-inducible immediate-early protein, CYR61, that promotes proliferation, migration, and adhesion, which was known to be similarly up-regulated by TGFBR1 and ACVR1 (19).

Furthermore, the current study revealed a number of genes that have not previously been implicated in TGFBR2 signaling. For example, up-regulation by wt-ACVR2 was observed for the proapoptotic genes NR4A1, DUSP2, and TNFRSF10C in addition to down-regulation of the antiapoptotic gene BIRC5. Increased expression of negative regulators of cell proliferation, including BTG1, PMP22, and TOB2 after wt-ACVR2 restoration was observed as well. Additionally, the following genes were also up-regulated by wt-ACVR2 transfection: MAPK6, a gene activated in response to growth factors through protein phosphorylation; HGS, a negative regulator of cell proliferation that undergoes tyrosine phosphorylation in response to epidermal growth factor and platelet-derived growth factor; and PPAP2B, a growth control gene that is known to be enhanced by epidermal growth factor in HeLa cells (24, 25, 26).

Recently, significant progress has been made toward identifying signal transduction pathways activated by TGF-β receptor family members. However, fewer studies have been published regarding the downstream effectors of activin receptors. In the present study, we focused our attention on ACVR2-regulated genes. We combined examination of the SMAD-mediated signaling pathway with gene expression profiling. Among genes identified as influenced by ACVR2 restoration, some had previously been related to TGF-β receptor signaling, whereas others were not previously suspected in either the ACVR2 or TGFβR2 pathways. The observed strong similarity between the activin and TGF-β signaling systems suggests that activin serves as an alternative activator of downstream TGF-β effectors in addition to SMADs. In addition, we confirmed that restoration of wt-ACVR2 function resulted in decreased cell growth and increased SMAD2 phosphorylation in a MSI-H colon cancer cell line with native biallelic ACVR2 frameshift mutation. Taken together, these data suggest that activin may serve as an alternative activator of SMADs and other downstream TGF-β effectors and that frameshift mutation of ACVR2 may contribute to MSI-H colon tumorigenesis via disruption of alternate TGF-β effector pathways.

Fig. 1.

ACVR2 expression analyses in transfected versus untransfected cells. A, ACVR2 mRNA levels in transfected cells. A1-A10: wt-ACVR2 transfected cells; C1-C8: positive control vector-transfected cells. Real-time quantitative RT-PCR analysis of ACVR2 mRNA expression levels in 10 wt-ACVR2- and 8 positive control vector-transfected clones revealed significantly higher levels of ACVR2 mRNA in wt-ACVR2 transfected cells (A1-A10) than in controls. Clones A3, A9, and A10 (exhibiting the highest levels of ACVR2 mRNA expression) as well as control clones C1, C2, and C3 were chosen for microarray study. B, ACVR2 protein levels in transfected cells. Western blotting analysis of protein lysates was done with a specific anti-ACVR2 antibody. A higher level of ACVR2 expression was seen in all of the 10 wt-ACVR2-transfected clones (Lanes 1–10) than in control vector-transfected cells (Lane B). Recombinant human ACVR2 protein (Sigma-Aldrich, St. Louis, MO) was used as a positive control (Lane A). The blot was then stripped with Restore Western Blot Stripping Buffer (Pierce) and reprobed with antiactin antibodies (Santa Cruz Biotechnology Biotech; bottom row).

Fig. 1.

ACVR2 expression analyses in transfected versus untransfected cells. A, ACVR2 mRNA levels in transfected cells. A1-A10: wt-ACVR2 transfected cells; C1-C8: positive control vector-transfected cells. Real-time quantitative RT-PCR analysis of ACVR2 mRNA expression levels in 10 wt-ACVR2- and 8 positive control vector-transfected clones revealed significantly higher levels of ACVR2 mRNA in wt-ACVR2 transfected cells (A1-A10) than in controls. Clones A3, A9, and A10 (exhibiting the highest levels of ACVR2 mRNA expression) as well as control clones C1, C2, and C3 were chosen for microarray study. B, ACVR2 protein levels in transfected cells. Western blotting analysis of protein lysates was done with a specific anti-ACVR2 antibody. A higher level of ACVR2 expression was seen in all of the 10 wt-ACVR2-transfected clones (Lanes 1–10) than in control vector-transfected cells (Lane B). Recombinant human ACVR2 protein (Sigma-Aldrich, St. Louis, MO) was used as a positive control (Lane A). The blot was then stripped with Restore Western Blot Stripping Buffer (Pierce) and reprobed with antiactin antibodies (Santa Cruz Biotechnology Biotech; bottom row).

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Fig. 2.

Cell growth assay of the wt-ACVR2 transfectants and control vector transfectants. This line graph displays results of direct cell counting over a 5-day period. The plots represent triplicate measurements for three wt-ACVR2 transfectants (▴) and three control vector transfectants (▪); bars, ±SD. The wt-ACVR2 transfectants showed significantly slower growth compared with control transfectants (*, measurement with P < 0.05, Student’s t test).

Fig. 2.

Cell growth assay of the wt-ACVR2 transfectants and control vector transfectants. This line graph displays results of direct cell counting over a 5-day period. The plots represent triplicate measurements for three wt-ACVR2 transfectants (▴) and three control vector transfectants (▪); bars, ±SD. The wt-ACVR2 transfectants showed significantly slower growth compared with control transfectants (*, measurement with P < 0.05, Student’s t test).

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Fig. 3.

SMAD2 protein expression and phosphorylation analyses in HCT15 colon cancer cells. A, SMAD2 and p-SMAD2 protein expression in wt-ACVR2 vector-transfected and untransfected cells. Immunoblotting was done with anti-SMAD2/3 and anti-p–SMAD2 antibodies. Total SMAD2 protein levels were equal in untransfected (top row, Lanes 1 and 2) and wt-ACVR2-transfected (top row, Lanes 3 and 4) HCT-15 cells. However, levels of p-SMAD2 protein were higher in wt-ACVR2-transfected cells (bottom row, Lanes 3 and 4) than in untransfected cells (bottom row, Lanes 1 and 2). This finding suggests that ACVR2 frameshift mutation results in impaired phosphorylation of SMAD2 proteins. B, activin-induced SMAD2 phosphorylation in wt-ACVR2 transfectants and control vector transfectants. Protein extracts were obtained from untreated (time 0) and from activin-treated cells after 1 and 2 hours of stimulation (time 1 and 2). Increased levels of p-SMAD2 were detected in wt-ACVR2 transfectants with the highest level after 2 hours of activin treatment, whereas no p-SMAD2 was detected in the control vector transfectants. HeLa cells untreated and treated with 100 ng of TGF-β1 were used as a positive control. Immunoblotting to anti-SMAD2 antibodies was done on the same membrane as anti-p–SMAD2 antibody immunoblotting after stripping of the previous antibody. Total SMAD2 protein levels were identical in both untreated and activin treated wt-ACVR2 transfectants as well as in control vector transfectants. The positions of signals detected by anti-p–SMAD2 and anti-SMAD2 antibodies were identical, additionally verifying the identity of the protein detected by anti-p–SMAD2 antibody.

Fig. 3.

SMAD2 protein expression and phosphorylation analyses in HCT15 colon cancer cells. A, SMAD2 and p-SMAD2 protein expression in wt-ACVR2 vector-transfected and untransfected cells. Immunoblotting was done with anti-SMAD2/3 and anti-p–SMAD2 antibodies. Total SMAD2 protein levels were equal in untransfected (top row, Lanes 1 and 2) and wt-ACVR2-transfected (top row, Lanes 3 and 4) HCT-15 cells. However, levels of p-SMAD2 protein were higher in wt-ACVR2-transfected cells (bottom row, Lanes 3 and 4) than in untransfected cells (bottom row, Lanes 1 and 2). This finding suggests that ACVR2 frameshift mutation results in impaired phosphorylation of SMAD2 proteins. B, activin-induced SMAD2 phosphorylation in wt-ACVR2 transfectants and control vector transfectants. Protein extracts were obtained from untreated (time 0) and from activin-treated cells after 1 and 2 hours of stimulation (time 1 and 2). Increased levels of p-SMAD2 were detected in wt-ACVR2 transfectants with the highest level after 2 hours of activin treatment, whereas no p-SMAD2 was detected in the control vector transfectants. HeLa cells untreated and treated with 100 ng of TGF-β1 were used as a positive control. Immunoblotting to anti-SMAD2 antibodies was done on the same membrane as anti-p–SMAD2 antibody immunoblotting after stripping of the previous antibody. Total SMAD2 protein levels were identical in both untreated and activin treated wt-ACVR2 transfectants as well as in control vector transfectants. The positions of signals detected by anti-p–SMAD2 and anti-SMAD2 antibodies were identical, additionally verifying the identity of the protein detected by anti-p–SMAD2 antibody.

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Grant support: CA85069, CA77057, CA95323, CA001808, CA098450, and the Medical Research Office, Department of Veterans Affairs.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: E. Deacu and Y. Mori contributed equally to this work. Supplementary data for this article can be found at Cancer Research Online at http://cancerres.aacrjournals.org.

Requests for reprints: Stephen J. Meltzer, University of Maryland School of Medicine, Division of Gastroenterology, Bressler Research Building, Room 8-009, 655 West Baltimore Street, Baltimore, MD 21201. Phone: (410) 706-3375; Fax: (410) 706-1099; E-mail: smeltzer@medicine.umaryland.edu

Table 1

Genes upregulated in wt-ACVR2 transfectants classified according to their involvement in biological processes

Gene symbolDescriptionAccession no.SAM scoreFold change
ACVR2 Activin type II receptor NM001616 8.47 160.93 
Apoptosis     
PEA15 Phosphoprotein enriched in astrocytes 15 NM003768 3.51 2.42 
TNFRSF10C Decoy receptor 1 (DcR1) NM003841 2.94 3.08 
DUSP2 Dual specificity phosphatase 2/PAC1 NM004418 4.06 9.96 
NR4A1 NAK1 DNA binding protein NM002135 4.68 2.52 
CyCS Cytochrome c NM018947 2.76 2.14 
SGK Serine/threonine protein kinase sgk NM005627 2.52 1.72 
Regulation of transcription     
JARID1C SMC (mouse) homolog, X chromosome (SMCX) NM004187 4.47 1.69 
JUN V-jun avian sarcoma virus 17 oncogene homolog (JUN) NM002228 3.76 5.26 
JUND Jun D proto-oncogene NM005354 6.42 2.82 
FOSB FBJ murine osteosarcoma viral oncogene homolog B NM006732 3.26 5.82 
KIAA0551 TRAF2 and NCK interacting kinase AB011123 3.97 1.64 
TBX2 T-box 2 NM005994 3.37 1.85 
ATF3 Activating transcription factor 3 NM001674 2.64 2.95 
EGR1 Early growth response 1 (EGR1) NM001964 2.44 2.41 
VGLL1 TONDU (TONDU)/vestigial like 1 (Drosophila) NM016267 2.40 2.31 
JUNB Jun B proto-oncogene NM002229 2.40 1.79 
CEBPA CCAAT/enhancer binding protein (C/EBP), α NM004364 2.39 2.42 
MSX1 Msh (Drosophila) homeo box homolog 1 NM002448 2.62 1.88 
HCA58 Hepatocellular carcinoma-associated antigen 58 AF220416 2.43 2.15 
RELA V-rel avian reticuloendotheliosis viral oncogene homolog A L19067 2.36 1.70 
IRF1 Interferon regulatory factor 1 NM002198 2.23 1.61 
TLE3 Transducin-like enhancer protein 3/KIAA1547 protein NM005078 3.15 1.96 
Cell growth and/or maintenance     
RHOB Ras homolog gene family, member B NM004040 3.86 3.91 
CYR61 Cysteine-rich, angiogenic inducer, 61 NM001554 6.21 6.5 
GPC1 Glypican 1 NM002081 6.21 2.10 
EXTL1 Tumor suppressor EXT-like protein NM001440 3.59 2.00 
NFKB2 Nuclear factor of κ light polypeptide gene enhancer in B-cells 2 (p49/p100) NM002502 3.04 2.04 
PPAP2B Phosphatidic acid phosphatase 2b NM003713 3.99 2.02 
JUN V-jun avian sarcoma virus 17 oncogene homolog NM002228 3.76 5.26 
INHA Inhibin A NM002191 3.32 1.81 
SLC3A2 Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 NM002394 2.29 2.32 
Signal transduction     
ARHE Ras homolog gene family, member E (RhoE) NM005168 3.27 2.54 
MAPK6 Mitogen-activated protein kinase 6/ERK3 NM002748 3.46 1.70 
LTB Lymphotoxin β NM002341 3.55 3.09 
EDN1 Endothelin 1 NM001955 3.51 2.53 
PPP2R2C Protein phosphatase 2, regulatory subunit B γ isoform XM029744 2.76 1.61 
CAPN5 Calpain5 NM004055 2.54 1.81 
MAP2K3 MAP kinase kinase 3b NM145110 2.38 2.10 
PLCB4 Phospholipase C, β 4 NM000933 2.62 1.91 
RAB6A RAB6A, member RAS oncogene family NM002869 2.58 1.52 
NR2F1 Nuclear receptor subfamily 2, group F, member 1 NM005654 2.48 1.75 
TBL3 Transducin β-like 3 NM006453 2.29 1.87 
PPP1R3A Protein phosphatase 1, regulatory (inhibitor) subunit 3A XM054777 2.24 1.68 
Cell adhesion     
ARHGDIA Rho GDP dissociation inhibitor α (RhoDHIA) NM004309 4.12 3.20 
CLSTN1 KIAA0911 protein NM014944 3.72 2.00 
LAMP2 Lysosomal-associated membrane protein 2 NM002294 2.98 2.44 
PVRL3 Nectin 3 NM015480 2.72 2.41 
PVRL2 Poliovirus receptor-related 2 (herpes virus entry mediator B) NM002856 2.66 2.39 
ATP2A2 ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 NM001681 2.36 1.58 
MLLT4 Myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, 4 NM005936 2.33 1.97 
VEL1 Vertebrate LIN7 homolog 1, Tax interaction protein 33 NM004664 2.88 2.54 
Regulation of cell proliferation     
BTG1 B-cell translocation gene 1, antiproliferative NM001731 3.58 2.02 
PMP22 Peripheral myelin protein 22 NM000304 3.52 2.01 
HGS Hepatocyte growth factor-regulated tyrosine kinase substrate NM004712 3.06 1.84 
LRPAP1 Low density lipoprotein-related protein-associated protein 1 NM002337 3.60 1.96 
IGF2 Insulin-like growth factor 2 (somatomedin A) NM000612 3.09 3.22 
DYRK1A Serine-threonine protein kinase (MNBH) NM101395 2.49 1.91 
TOB2 Transducer of ERBB2, 2 NM016272 2.37 1.84 
Cell-cell signaling     
GIP2 Interferon-stimulated protein, 15 kDa (ISG15) NM005101 2.78 2.90 
Gene symbolDescriptionAccession no.SAM scoreFold change
ACVR2 Activin type II receptor NM001616 8.47 160.93 
Apoptosis     
PEA15 Phosphoprotein enriched in astrocytes 15 NM003768 3.51 2.42 
TNFRSF10C Decoy receptor 1 (DcR1) NM003841 2.94 3.08 
DUSP2 Dual specificity phosphatase 2/PAC1 NM004418 4.06 9.96 
NR4A1 NAK1 DNA binding protein NM002135 4.68 2.52 
CyCS Cytochrome c NM018947 2.76 2.14 
SGK Serine/threonine protein kinase sgk NM005627 2.52 1.72 
Regulation of transcription     
JARID1C SMC (mouse) homolog, X chromosome (SMCX) NM004187 4.47 1.69 
JUN V-jun avian sarcoma virus 17 oncogene homolog (JUN) NM002228 3.76 5.26 
JUND Jun D proto-oncogene NM005354 6.42 2.82 
FOSB FBJ murine osteosarcoma viral oncogene homolog B NM006732 3.26 5.82 
KIAA0551 TRAF2 and NCK interacting kinase AB011123 3.97 1.64 
TBX2 T-box 2 NM005994 3.37 1.85 
ATF3 Activating transcription factor 3 NM001674 2.64 2.95 
EGR1 Early growth response 1 (EGR1) NM001964 2.44 2.41 
VGLL1 TONDU (TONDU)/vestigial like 1 (Drosophila) NM016267 2.40 2.31 
JUNB Jun B proto-oncogene NM002229 2.40 1.79 
CEBPA CCAAT/enhancer binding protein (C/EBP), α NM004364 2.39 2.42 
MSX1 Msh (Drosophila) homeo box homolog 1 NM002448 2.62 1.88 
HCA58 Hepatocellular carcinoma-associated antigen 58 AF220416 2.43 2.15 
RELA V-rel avian reticuloendotheliosis viral oncogene homolog A L19067 2.36 1.70 
IRF1 Interferon regulatory factor 1 NM002198 2.23 1.61 
TLE3 Transducin-like enhancer protein 3/KIAA1547 protein NM005078 3.15 1.96 
Cell growth and/or maintenance     
RHOB Ras homolog gene family, member B NM004040 3.86 3.91 
CYR61 Cysteine-rich, angiogenic inducer, 61 NM001554 6.21 6.5 
GPC1 Glypican 1 NM002081 6.21 2.10 
EXTL1 Tumor suppressor EXT-like protein NM001440 3.59 2.00 
NFKB2 Nuclear factor of κ light polypeptide gene enhancer in B-cells 2 (p49/p100) NM002502 3.04 2.04 
PPAP2B Phosphatidic acid phosphatase 2b NM003713 3.99 2.02 
JUN V-jun avian sarcoma virus 17 oncogene homolog NM002228 3.76 5.26 
INHA Inhibin A NM002191 3.32 1.81 
SLC3A2 Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 NM002394 2.29 2.32 
Signal transduction     
ARHE Ras homolog gene family, member E (RhoE) NM005168 3.27 2.54 
MAPK6 Mitogen-activated protein kinase 6/ERK3 NM002748 3.46 1.70 
LTB Lymphotoxin β NM002341 3.55 3.09 
EDN1 Endothelin 1 NM001955 3.51 2.53 
PPP2R2C Protein phosphatase 2, regulatory subunit B γ isoform XM029744 2.76 1.61 
CAPN5 Calpain5 NM004055 2.54 1.81 
MAP2K3 MAP kinase kinase 3b NM145110 2.38 2.10 
PLCB4 Phospholipase C, β 4 NM000933 2.62 1.91 
RAB6A RAB6A, member RAS oncogene family NM002869 2.58 1.52 
NR2F1 Nuclear receptor subfamily 2, group F, member 1 NM005654 2.48 1.75 
TBL3 Transducin β-like 3 NM006453 2.29 1.87 
PPP1R3A Protein phosphatase 1, regulatory (inhibitor) subunit 3A XM054777 2.24 1.68 
Cell adhesion     
ARHGDIA Rho GDP dissociation inhibitor α (RhoDHIA) NM004309 4.12 3.20 
CLSTN1 KIAA0911 protein NM014944 3.72 2.00 
LAMP2 Lysosomal-associated membrane protein 2 NM002294 2.98 2.44 
PVRL3 Nectin 3 NM015480 2.72 2.41 
PVRL2 Poliovirus receptor-related 2 (herpes virus entry mediator B) NM002856 2.66 2.39 
ATP2A2 ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 NM001681 2.36 1.58 
MLLT4 Myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, 4 NM005936 2.33 1.97 
VEL1 Vertebrate LIN7 homolog 1, Tax interaction protein 33 NM004664 2.88 2.54 
Regulation of cell proliferation     
BTG1 B-cell translocation gene 1, antiproliferative NM001731 3.58 2.02 
PMP22 Peripheral myelin protein 22 NM000304 3.52 2.01 
HGS Hepatocyte growth factor-regulated tyrosine kinase substrate NM004712 3.06 1.84 
LRPAP1 Low density lipoprotein-related protein-associated protein 1 NM002337 3.60 1.96 
IGF2 Insulin-like growth factor 2 (somatomedin A) NM000612 3.09 3.22 
DYRK1A Serine-threonine protein kinase (MNBH) NM101395 2.49 1.91 
TOB2 Transducer of ERBB2, 2 NM016272 2.37 1.84 
Cell-cell signaling     
GIP2 Interferon-stimulated protein, 15 kDa (ISG15) NM005101 2.78 2.90 
Table 1A

Continued

Gene symbolDescriptionAccession no.SAM scoreFold change
RNA processing     
CHERP Protein with polyglutamine repeat; calcium (ca2+) homeostasis endoplasmic reticulum protein NM006387 5.98 2.92 
MPHOSPH10 M-phase phosphoprotein 10 NM005791 3.09 1.76 
SLBP Hairpin binding protein, histone (HBP) NM006527 2.50 2.25 
Intercellular junction assembly     
GJA7 Gap junction protein, α 7, 45kD (connexin 45) NM005497 2.54 2.39 
Transport     
ATP7B ATPase, Cu++ transporting, β polypeptide (Wilson disease) NM000053 4.10 6.14 
ATP6IP1 ATPase, H+ transporting, lysosomal, subunit 1 NM001183 3.76 1.80 
SLC16A1 Monocarboxylate transporter 1 (SLC16A1) NM003051 3.07 1.65 
ATP6V0C ATPase, H+ transporting, lysosomal (vacuolar proton pump) NM001694 3.04 1.50 
GTF3C1 General transcription factor IIIC, polypeptide 1 (α subunit) NM001520 3.90 1.83 
TLOC1 Translocation protein 1 NM003262 3.13 2.17 
NAPG N-ethylmaleimide-sensitive factor attachment protein, γ NM003826 2.29 2.32 
Metabolism     
ALDH1A1 Aldehyde dehydrogenase 1 family, member A1 NM000689 3.89 5.20 
AUP1 Ancient ubiquitous protein 1 AF100754 3.71 1.72 
ATP1A1 ATPase, Na+/K+ transporting, α 1 polypeptide NM000701 3.28 1.50 
AGPAT2 Lysophosphatidic acid acyltransferase NM006412 2.52 3.07 
RNA binding     
RBM12 Putative brain nuclearly-targeted protein AB018308 3.77 1.96 
EIF2S1 Translation initiation factor eIF-2α NM004094 2.93 1.67 
FMR1 Fragile X mental retardation 1 NM002024 2.32 2.06 
Protein modification     
PLOD3 Procollagen-lysine 2-oxoglutarate 5-dioxygenase NM001084 3.66 2.35 
PTPRU Receptor protein tyrosine phosphatase hPTP-J precursor NM005704 3.37 1.73 
Proteolysis and peptidolysis     
WFDC2 WAP four-disulfide core domain 2 NM006103 2.54 2.49 
C1S Complement component 1, s subcomponent NM001734 2.50 3.30 
PSMD13 Proteasome (prosome, macropain) 26S subunit NM002817 2.62 1.66 
PRSS8 Prostasin NM002773 2.40 1.73 
Cytoskeletal anchoring     
VIL2 Vilin2 NM003379 3.11 2.07 
SDC3 Syndecan 3 (N-syndecan) AB007937 2.26 2.04 
PLEC1 Plectin 1 NM000445 2.25 1.75 
Regulation of cell migration     
SERPINE2 Plasminogen activator inhibitor type 1 member NM006216 2.60 3.67 
Heat shock protein activity     
SERPINH2 Collagen binding protein 2 NM001235 2.98 2.08 
HSPH1 Heat shock Mr 105,000 (HSP105B) NM006644 2.85 3.65 
DNAJB1 Heat shock Mr 40,000 protein 1 (HSPF1) NM006145 3.10 2.19 
HSPA6 Heat shock Mr 70,000 protein 6 (HSP70B′) NM002155 2.49 2.29 
Defense response     
TFF3 Trefoil factor 3 (intestinal) NM003226 2.51 2.51 
Nucleosome assembly     
HIST1H1C H1 histone family, member 2 (H1F2) NM005319 2.47 3.62 
Others     
HMOX1 Heme oxygenase (decycling) 1 NM002133 2.41 2.42 
CDR2 Cerebellar degeneration-related protein 2 BC017503 2.97 2.20 
A2LP Ataxin 2 related protein NM007245 2.94 2.04 
PCOLN3 Procollagen (type III) N-endopeptidase NM002768 2.83 1.55 
RNF13 Ring finger protein 13 NM007282 2.43 1.96 
IER2 Immediate early protein (ETR101) NM004907 2.91 2.80 
HCA58 Hepatocellular carcinoma-associated antigen 58 AF220416 2.43 2.15 
Gene symbolDescriptionAccession no.SAM scoreFold change
RNA processing     
CHERP Protein with polyglutamine repeat; calcium (ca2+) homeostasis endoplasmic reticulum protein NM006387 5.98 2.92 
MPHOSPH10 M-phase phosphoprotein 10 NM005791 3.09 1.76 
SLBP Hairpin binding protein, histone (HBP) NM006527 2.50 2.25 
Intercellular junction assembly     
GJA7 Gap junction protein, α 7, 45kD (connexin 45) NM005497 2.54 2.39 
Transport     
ATP7B ATPase, Cu++ transporting, β polypeptide (Wilson disease) NM000053 4.10 6.14 
ATP6IP1 ATPase, H+ transporting, lysosomal, subunit 1 NM001183 3.76 1.80 
SLC16A1 Monocarboxylate transporter 1 (SLC16A1) NM003051 3.07 1.65 
ATP6V0C ATPase, H+ transporting, lysosomal (vacuolar proton pump) NM001694 3.04 1.50 
GTF3C1 General transcription factor IIIC, polypeptide 1 (α subunit) NM001520 3.90 1.83 
TLOC1 Translocation protein 1 NM003262 3.13 2.17 
NAPG N-ethylmaleimide-sensitive factor attachment protein, γ NM003826 2.29 2.32 
Metabolism     
ALDH1A1 Aldehyde dehydrogenase 1 family, member A1 NM000689 3.89 5.20 
AUP1 Ancient ubiquitous protein 1 AF100754 3.71 1.72 
ATP1A1 ATPase, Na+/K+ transporting, α 1 polypeptide NM000701 3.28 1.50 
AGPAT2 Lysophosphatidic acid acyltransferase NM006412 2.52 3.07 
RNA binding     
RBM12 Putative brain nuclearly-targeted protein AB018308 3.77 1.96 
EIF2S1 Translation initiation factor eIF-2α NM004094 2.93 1.67 
FMR1 Fragile X mental retardation 1 NM002024 2.32 2.06 
Protein modification     
PLOD3 Procollagen-lysine 2-oxoglutarate 5-dioxygenase NM001084 3.66 2.35 
PTPRU Receptor protein tyrosine phosphatase hPTP-J precursor NM005704 3.37 1.73 
Proteolysis and peptidolysis     
WFDC2 WAP four-disulfide core domain 2 NM006103 2.54 2.49 
C1S Complement component 1, s subcomponent NM001734 2.50 3.30 
PSMD13 Proteasome (prosome, macropain) 26S subunit NM002817 2.62 1.66 
PRSS8 Prostasin NM002773 2.40 1.73 
Cytoskeletal anchoring     
VIL2 Vilin2 NM003379 3.11 2.07 
SDC3 Syndecan 3 (N-syndecan) AB007937 2.26 2.04 
PLEC1 Plectin 1 NM000445 2.25 1.75 
Regulation of cell migration     
SERPINE2 Plasminogen activator inhibitor type 1 member NM006216 2.60 3.67 
Heat shock protein activity     
SERPINH2 Collagen binding protein 2 NM001235 2.98 2.08 
HSPH1 Heat shock Mr 105,000 (HSP105B) NM006644 2.85 3.65 
DNAJB1 Heat shock Mr 40,000 protein 1 (HSPF1) NM006145 3.10 2.19 
HSPA6 Heat shock Mr 70,000 protein 6 (HSP70B′) NM002155 2.49 2.29 
Defense response     
TFF3 Trefoil factor 3 (intestinal) NM003226 2.51 2.51 
Nucleosome assembly     
HIST1H1C H1 histone family, member 2 (H1F2) NM005319 2.47 3.62 
Others     
HMOX1 Heme oxygenase (decycling) 1 NM002133 2.41 2.42 
CDR2 Cerebellar degeneration-related protein 2 BC017503 2.97 2.20 
A2LP Ataxin 2 related protein NM007245 2.94 2.04 
PCOLN3 Procollagen (type III) N-endopeptidase NM002768 2.83 1.55 
RNF13 Ring finger protein 13 NM007282 2.43 1.96 
IER2 Immediate early protein (ETR101) NM004907 2.91 2.80 
HCA58 Hepatocellular carcinoma-associated antigen 58 AF220416 2.43 2.15 

NOTE. Fold change is the average of the relative expression ratios for the three wt-ACVR2-transfectants to the reference probe divided by the average of the relative expression ratios for the three vector control transfectants to the reference probe. Bold represents genes previously reported by various studies as related to TGF-β signaling pathway (16171819, 23).

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