Expression of SnoN is elevated in human cancer cells.
To understand the contribution of SnoN and Ski to epithelial cell transformation, we first examined their expression levels in a number of cell lines derived from different types of human cancer. These included human breast cancer cell lines (MDA-MB-231, MDA-MB-435, and ZR75b), lung adenocarcinoma lines (A427, A549, and SW1271), melanoma (A375), and osteosarcoma (HT1080). Two untransformed human mammary epithelial cell lines, HMT3522-S1 and MCF10A, were included as normal controls. Endogenous SnoN and Ski proteins were isolated by immunoprecipitation from these cell lines, and their expression was detected by immunoblotting with anti-SnoN and anti-Ski antisera, respectively. The level of SnoN protein was strongly elevated in all cancer lines examined compared to untransformed cells, which express low or undetectable levels of SnoN (Fig. ). This upregulation of SnoN is mostly due to an increase in snoN mRNA (Fig. and data not shown). In addition, no mutations were detected in endogenous SnoN isolated from some of the cancer cell lines. In contrast, the related Ski protein was detected readily in nontumorigenic cell lines, and its expression was barely detectable in some human cancer cell lines, particularly the A549 lung adenocarcinoma and A375 melanoma cells. No significant correlation was observed between SnoN or Ski expression and the intensity and duration of TGF-β-induced phosphorylation of Smad2 and Smad3 (Fig. ), consistent with the notion that SnoN and Ski function downstream of Smad phosphorylation.
FIG. 1. Expression of SnoN is elevated in human cancer cell lines. (A) Expression levels of SnoN and Ski are regulated differently in human cancer cell lines. Endogenous SnoN or Ski was isolated by immunoprecipitation from equal amounts of cell lysates prepared (more ...)
Thus, the expression of SnoN and Ski appears to be regulated differently during malignant progression. Since SnoN expression is elevated in all cancer cell lines we surveyed, we decided to focus on the role of SnoN in various aspects of mammalian tumorigenesis in this study.
Downregulation of SnoN expression restores TGF-β responses.
We chose two model systems, the A549 human lung adenocarcinoma cell line and the MDA-MB-231 human breast cancer cell line, to investigate the role of SnoN in malignant progression. A549 cells express high levels of SnoN but have very little if any Ski (Fig. ). MDA-MB-231 cells are derived from invasive and metastatic breast adenocarcinoma and express both SnoN and Ski at high levels. These cell lines are weakly or nonresponsive to TGF-β-induced growth inhibition and, in addition to various aspects of morphological and mitogenic transformation, are also capable of undergoing or have undergone at least some aspects of EMT, an important process in later stages of tumorigenesis that may be necessary for tumor invasiveness and metastasis. The multiple features of tumorigenesis exhibited by these cell lines provide an opportunity to probe the contribution of SnoN to various aspects of epithelial transformation. In addition, the different tissue origins of the two cell lines allow us to determine whether the processes affected by SnoN are tissue specific or are common to epithelial tumors.
We generated stable A549 and MDA-MB-231 cell lines in which expression of SnoN was reduced using shRNA specific for human snoN. pSUPER vector expressing snoN shRNA was introduced into A549 and MDA-MB-231 cells by transfection together with a plasmid expressing a puromycin resistance gene. For each cell line, multiple stable clones were generated that exhibited reduced expression of SnoN (Fig. ). Stable reduction of SnoN expression had no effect on the intensity or duration of TGF-β-induced phosphorylation of Smad2 or Smad3, as expected given that SnoN plays no direct role in the regulation of Smad phosphorylation (Fig. ).
FIG. 2. Reduction of SnoN expression restores TGF-β responses in A549 and MDA-MB-231 cancer cell lines. (A) Reduction of snoN expression in A549 lung cancer cells and MDA-MB-231 breast cancer cells by shRNA. Stable shSnoN cell lines expressing the shRNA (more ...)
We have shown previously that SnoN represses TGF-β signaling (56
). Therefore, reducing SnoN expression in these TGF-β-unresponsive cancer cell lines might relieve this repression and restore TGF-β signaling. Indeed, in a growth inhibition assay, while the parental A549 cell line was only weakly responsive to TGF-β, cells expressing shSnoN showed a markedly enhanced response to TGF-β-induced growth inhibition (Fig. , left panel). MDA-MB-231 cells have completely lost any growth arrest response to TGF-β and instead proliferate in the presence of TGF-β (Fig. , right panel). Reducing SnoN expression blocked this increased proliferation but did not inhibit the growth of MDA-MB-231 cells (Fig. , right panel). Interestingly, reduction of both SnoN and Ski expression in MDA-MB-231 cells permitted a moderate growth arrest response, with 32% growth inhibition occurring at 200 pM TGF-β, suggesting that the high level of Ski expression in MDA-MB-231 cells may also contribute to the repression of TGF-β-elicited growth arrest.
We next examined whether reducing SnoN expression augmented activation of endogenous TGF-β target gene expression (Fig. ). In parental A549 and MDA-MB-231 cells, TGF-β induced expression of PAI-1 mRNA after 3 h of treatment (Fig. , left panel). This induction of PAI-1 by TGF-β was enhanced in both A549 and MDA-MB-231 cells expressing snoN shRNA (Fig. , left panel). Likewise, the expression of p21CIP1, another TGF-β-inducible gene, was elevated in shSnoN A549 cells even in the absence of TGF-β treatment and further enhanced by TGF-β stimulation (Fig. , right panel). This elevation in basal-level p21 expression in shSnoN A549 cells is not due to the increased autocrine TGF-β activity in these cells, because inhibition of ΤβRI activity by an inhibitor, SB431542, had no effect on the basal expression of p21, even though this treatment readily blocked TGF-β-induced p21 expression (data not shown). This observation is also consistent with the proposed role of SnoN in maintaining the basal states of some TGF-β-responsive genes. Taken together, these results suggest that reducing SnoN expression in human lung and breast cancer cells enhances cellular responses to TGF-β.
Reducing SnoN expression suppresses tumor growth.
TGF-β signaling suppresses tumor growth at early stages of tumorigenesis through its ability to elicit growth arrest (22
). Since SnoN represses TGF-β signaling, we reasoned that reducing SnoN expression in cancer cells might diminish or reverse their transformed phenotype. To test this, we first examined the ability of shSnoN-expressing lung and breast cancer cells to undergo anchorage-independent growth in a soft agar assay. Cells were embedded in soft agar and allowed to form colonies for approximately 3 weeks. Under these conditions, parental A549 and MDA-MB-231 cell lines formed colonies readily, whereas cells with reduced expression of SnoN were severely impaired in their growth in soft agar (Fig. ). Interestingly, Ski does not appear to play a major role in anchorage-independent growth, since shSki-expressing MDA-MB-231 cells have significantly reduced expression levels of Ski protein yet are still able to form as many soft agar colonies as parental cells do (Fig. , bottom panel). Furthermore, reducing Ski expression in shSnoN-expressing cells did not result in further reduction in soft agar colony formation (data not shown). These results suggest that SnoN but not Ski functions to promote mitogenic transformation.
FIG. 3. Reducing SnoN expression suppresses the transformed phenotype of cancer cells. (A) Reducing SnoN expression reverses transformation of A549 and MDA-MB-231 cancer cells. Parental A549 or MDA-MB-231 cells and their derived shSnoN cells were subjected to (more ...)
We next examined whether the decrease in the transforming activity of SnoN-deficient cells in culture resulted in reduced tumorigenicity in vivo. Nude mice were injected with parental cancer cell lines or those lacking SnoN, and tumor number and volume were measured after 8 weeks. Reduction of SnoN expression in lung cancer cells led to a significant decrease in tumor formation, with 40% of shSnoN cell injections leading to tumor formation, compared with 100% of parental A549 cell injections forming tumors (Table ). Of those SnoN-deficient cells that formed tumors, the average tumor volume was significantly smaller than those formed by parental A549 cells (Fig. and Table ). Similarly, in nude mice injected with shSnoN-expressing MDA-MB-231 breast cancer cells, a modest but reproducible decrease in tumor number was observed, consistent with the more aggressive and advanced nature of these malignant cells compared with A549 cells (Table ). As with shSnoN-expressing A549 cells, the tumors that formed from SnoN-deficient MDA-MB-231 cells were significantly smaller (Table ).
Tumorigenicity in athymic nude micea
Taken together, our results indicate that SnoN has prooncogenic activity and functions to promote tumor growth both in vitro and in vivo.
Downregulation of SnoN enhances TGF-β-induced EMT.
The EMT is a process by which tumor cells lose epithelial characteristics and acquire the features of mesenchymal cells. It is believed to promote the ability to invade surrounding tissues and blood vessels and undergo metastasis and is thought to be important for malignant progression in vivo. EMT is characterized by a number of morphological and biochemical changes, including increased cell motility and stress fiber formation, downregulation of adherens junctions and their affiliated proteins, including E-cadherin, induction of extracellular matrix (ECM) proteins, and increased MMP activity (66
). Since SnoN potentiates oncogenic transformation and tumor growth, we next asked whether and how SnoN affects the EMT using the A549 and MDA-MB-231 cell lines expressing shSnoN.
We first examined whether expression of SnoN affects the motility of A549 and MDA-MB-231 cells in a wound healing assay. A wound was created by scratching a confluent monolayer of cells with a pipette tip, and relative rates of cell motility were assessed by measuring percent closure of the wound after 48 h of cell migration. Parental A549 cells showed only 18% wound closure within this time period, whereas migration of shSnoN A549 cells resulted in 74% wound closure (Fig. ), suggesting that reducing SnoN expression markedly increased cell motility. Similar results were obtained in a transwell migration assay (data not shown). Increased cell motility was also observed in SnoN-deficient MDA-MB-231 breast cancer cells (Fig. , right panel). Since the MDA-MB-231 cells already exhibit a high rate of cell migration, reducing SnoN expression only resulted in a moderate, but reproducible, increase in cell motility. Taken together, these data suggest that SnoN functions to repress cell motility.
FIG. 4. Reducing SnoN expression enhances EMT. (A) Reducing SnoN expression increases cell motility. Confluent cell monolayers of parental or shSnoN-expressing A549 and MDA-MB-231 cells were wounded with a pipette tip. Wound closure was monitored by microscopy (more ...)
Cells that have undergone the EMT also display increased ECM deposition as well as elevated activity of MMPs, for which ECM acts as a substrate. This dynamic production and degradation of ECM is thought to facilitate the movement of tumor cells during metastasis (27
). In order to test whether SnoN expression affects induction of ECM proteins, we examined the expression of fibronectin in parental and shSnoN-expressing A549 cells in the absence and presence of TGF-β. TGF-β treatment induced an increase in fibronectin expression in parental lung cancer cells (Fig. ). This increase was significantly enhanced in shSnoN cells, suggesting that SnoN normally inhibits induction of fibronectin by TGF-β (Fig. ). In situ zymography was employed to examine MMP activity in parental and shSnoN-expressing cells. Cells were plated on fluorescently labeled gelatin, which is a substrate for proteases such as MMP2 and MMP9. Protease activity was assessed by quantifying the degradation of the fluorescently labeled substrates (Fig. ). The protease activity was significantly increased in shSnoN-expressing cells relative to parental cells (11.1-fold and 7.5-fold increase in shSnoN A549 cells and MDA-MB-231 cells, respectively) (Fig. , right panels), and this activity was inhibited by the addition of the MMP inhibitor GM6001, suggesting that the protease activity observed was specific to MMPs (Fig. ). Treatment with TGF-β stimulated protease activity, as has been reported previously (1
), and this TGF-β-induced protease activity was also slightly enhanced in SnoN-deficient tumor cells.
Upon treatment with TGF-β, A549 cells exhibited morphological changes characteristic of EMT, becoming more scattered and elongated, and these TGF-β-induced changes in cell morphology were more pronounced in shSnoN cells (data not shown). Morphological changes occurring during EMT are thought to be due to increased actin stress fibers as well as loss of adherens junctions resulting from downregulation or mislocalization of E-cadherin (27
). We therefore examined whether the reduction of SnoN expression affected stress fiber formation by staining cells with fluorescently labeled phalloidin. In parental A549 lung cancer cells and MDA-MB-231 breast cancer cells, cellular actin was arranged cortically, with few or no stress fibers present (Fig. ). As has been demonstrated previously in several cell types (9
), TGF-β treatment resulted in increased stress fiber formation (Fig. ). In shSnoN-expressing lung and breast cancer cells, actin stress fibers were observed even in the absence of TGF-β, and stress fiber formation was further enhanced upon stimulation with TGF-β (Fig. ). Thus, downregulation of SnoN in both lung and breast cancer cells augmented actin stress fiber formation.
Loss of adherens junctions due to downregulation or mislocalization of E-cadherin is frequently observed as tumor cells progress to later, more invasive stages of carcinogenesis (49
). MDA-MB-231 cells have already lost E-cadherin expression, since they are quite advanced in malignant progression. We therefore examined expression of E-cadherin in parental and shSnoN-expressing A549 cells. TGF-β induced a very slight reduction in E-cadherin levels in parental A549 cells (Fig. ). Basal levels of E-cadherin were reduced in shSnoN cells compared with parental A549 cells, and this reduction was more pronounced upon treatment with TGF-β (Fig. ), suggesting that downregulation of SnoN promotes disruption of cell-cell contacts. These data are consistent with the morphological observation of enhanced EMT in shSnoN cells.
Taken together, these data indicate that in addition to prooncogenic activity, SnoN also possesses antitumorigenic activity by inhibiting the EMT and possibly tumor metastasis.
Downregulation of SnoN enhances tumor metastasis in vivo.
The EMT is thought to play a role in tumor cell metastasis (59
). To investigate how SnoN affects tumor cell metastasis, we tested the ability of MDA-MB-231 cells with reduced SnoN expression to form secondary bone and lung metastases in an in vivo metastasis mouse model system. Cells were injected into the left cardiac ventricle of nude mice, and osteolytic bone metastases were quantified after 4 weeks by the histomorphometric measurement of tumor area/burden (in percentage), and metastasis to lung was examined by anatomical analysis of fixed lung tissue at the end of 4 weeks. Interestingly, cells with reduced expression of SnoN appeared to have a moderate but reproducible increase in metastasis to both bone and lung than that of the parental breast cancer cells (Fig. ). The heightened skeletal metastatic tumor burden in mice inoculated with shSnoN-expressing cells also resulted in severe paraplegia, as evidenced by their diminished latency to fall from a wire hang test (Fig. ). Since the formation of metastatic colonies in the in vivo metastasis assay is dependent upon a number of cellular attributes acting in concert, including proliferative potential, migration, and invasion, and in light of the fact that shSnoN cells exhibited significantly reduced proliferative potential, the observation of increased skeletal and lung metastasis may in fact be an under-representation of the migratory and invasive potential of these cells.
FIG. 5. Effects of downregulation of SnoN on tumor metastasis in vivo. (A) Effects of abrogation of SnoN expression on the lung metastasis of MDA-MB-231 cells. Parental or shSnoN-expressing MDA-MB-231 cells were injected into the left cardiac ventricle of 4-week-old (more ...)
We also tested the effects of SnoN on the metastatic potential of lung cancer cells. In this assay, parental A549 lung cancer cells, or the shSnoN-expressing derivatives, were injected into the tail vein of nude mice. If these cells undergo metastasis, they will form secondary tumor modules in the lung that can be identified readily in fixed lung tissues. As shown in Fig. , injection of parental A549 cells resulted in the formation of extensive lung metastases. In contrast to the results observed in SnoN-deficient breast cancer cells, reducing the level of SnoN expression in lung cancer cells significantly reduced the formation of lung metastases (Fig. ). However, it is important to bear in mind that the outcome of these in vivo experiments depends on the combined effects of tumor growth and tumor metastasis. Since the growth of A549 cells is dramatically blocked by a reduction in SnoN expression (Table ), it is possible that the more pronounced reduction in tumor growth potential in lung cancer cells precluded their ability to form metastatic nodules. In order to bypass the interference of the growth effects and focus on the metastatic potential of these cells, we examined the expression of Twist1, a known marker of metastasis, in SnoN-deficient lung cancer cells. In A549 lung cancer cells expressing shSnoN, a modest but reproducible increase in Twist1 expression was detected (Fig. ). This elevation in Twist1 expression was also confirmed by microarray analysis (see Table and Fig. , below). These results are consistent with the increased EMT observed in shSnoN-expressing lung cancer cells and suggest a role for SnoN in inhibiting tumor metastasis.
Selected genes whose expression is altered upon reduction of SnoN expressiona
FIG. 8. Alterations in gene expression between parental and SnoN-deficient lung cancer cells. Changes in gene expression for seven loci (JunB, GADD45A, EGFR, Twist1, VEGF, PLAU, and EMP1) identified in Table were confirmed by RT-PCR. RT-PCR with (more ...) The effects of SnoN on malignant progression are mediated by both Smad-dependent and Smad-independent mechanisms.
In order to confirm the specificity of the results observed in shSnoN cells, we performed “rescue” experiments by reintroducing a wild-type SnoN (WTSnoN) back into the shSnoN-expressing A549 cells and asking whether the reexpressed SnoN reversed the phenotypes of the shSnoN cells. To determine whether the observed effects of SnoN on various aspects of tumorigenesis require its ability to interact with the Smad proteins, a mutant SnoN lacking Smad binding sites (mSnoN) (29
) was introduced back into the SnoN-deficient A549 lung cancer cells in parallel with WTSnoN. Both WTSnoN and mSnoN expression constructs contain silent mutations in the snoN
cDNA that blocks recognition by the snoN
shRNA. Stable A549 shSnoN clones expressing WTSnoN or mSnoN were generated, and expression of the SnoN proteins was confirmed by Western blotting (Fig. ). At least three clones of each were examined in all the biological and biochemical tests, and results from a representative clone are shown. In a growth inhibition assay, reexpression of WTSnoN markedly impaired the ability of shSnoN A549 cells to undergo growth inhibition in response to TGF-β (78% versus 40% growth inhibition, respectively) (Fig. ). In contrast, expression of mSnoN did not rescue TGF-β-induced growth arrest (Fig. ). This is as expected, since SnoN antagonizes TGF-β signaling through binding to the Smads.
FIG. 6. The effects of SnoN on tumorigenesis are mediated by both Smad-dependent and Smad-independent pathways. (A) Expression levels of wild-type SnoN (WTSnoN) and a mutant SnoN defective in Smad binding (mSnoN) in shSnoN-expressing A549 cells. WTSnoN or mSnoN (more ...)
We next tested the ability of WTSnoN to rescue the effects of reducing SnoN on various aspects of malignant transformation. Reexpression of SnoN in shSnoN A549 cells partially restored their ability to undergo anchorage-independent growth (Fig. ). The inability of reintroduced SnoN to fully restore oncogenic transformation is mostly likely due to the lower expression level of the ectopically expressed WTSnoN compared with endogenous SnoN in parental A549 cells, as shown in Fig. . Similarly, introduction of WTSnoN mitigated the enhanced EMT responses observed in shShoN-expressing lung cancer cells proportionate to its expression level, as evidenced by the reduced cell motility, the decrease in stress fiber formation, the increase in localization of E-cadherin at the cell junction, the decrease in TGF-β-induced fibronectin production, and the decrease in MMP2 activity (Fig. ). These data confirmed that the phenotypes observed in shSnoN cells are specifically due to the reduction of SnoN expression.
In contrast, introduction of the mutant SnoN defective in binding to the Smad proteins (mSnoN) back into the shSnoN cells failed to restore the anchorage-independent growth (Fig. ), even though the expression level of ectopically expressed mSnoN was higher than that of WTSnoN in rescued cells and was comparable to that of endogenous SnoN in parental A549 cells (Fig. ). Surprisingly and interestingly, mSnoN can rescue some of the EMT phenotypes found in shSnoN cells, including E-cadherin localization, fibronectin production, and MMP2 activity, but not others, such as stress fiber formation and cell motility (Fig. ). These data suggest that SnoN inhibits the EMT through both Smad-dependent and Smad-independent pathways and that both pathways are necessary for its effects on malignant progression.
SnoN inhibits RhoA GTPase activity to repress actin stress fiber formation.
Since SnoN can activate both Smad-dependent and Smad-independent pathways to regulate EMT, we began to dissect downstream signaling events that may mediate these activities of SnoN. Two approaches were taken. In the first approach, we examined signaling molecules known to regulate various aspects of cell growth, migration, and morphology. In the second approach, microarray analysis was carried out to compare the patterns of gene expression between parental and shSnoN-expressing A549 cells.
The Rho family of small GTPases, including RhoA, Rac, and Cdc42, play important roles in the regulation of cell growth, motility, and actin stress fiber formation (25
). Since reducing SnoN expression markedly enhanced actin stress fiber formation, we examined whether SnoN regulates the Rho family of proteins by comparing the expression and activity of RhoA, Rac, and Cdc42 in parental and shSnoN-expressing A549 cells. While SnoN expression had no effect on the expression levels of RhoA, Rac, or Cdc42, the GTP-binding activity of RhoA, but not that of Rac or Cdc42, was significantly elevated in SnoN-deficient cells (Fig. ). As reported before (9
), TGF-β stimulation resulted in a modest increase in RhoA activity, and this increase was further enhanced in shSnoN cells (Fig. ). To determine whether this increase in RhoA activity was required for the enhanced stress fiber formation observed in SnoN-deficient cells, a dominant negative RhoA (DNRhoA:RhoA T19N) was introduced into the shSnoN cells. In untransfected shSnoN A549 or shSnoN MDA-MB-231 cells, actin was arranged in elongated stress fibers as observed previously (Fig. and ). In contrast, cells expressing DNRhoA exhibited diffused cytoplasmic actin staining with no detectable stress fibers (Fig. ), suggesting that RhoA activity is required for the increased stress fiber formation observed in SnoN-deficient cells.
FIG. 7. SnoN inhibits RhoA GTPase activity to block stress fiber formation. (A) Activity of RhoA, not Rac1 or Cdc42, is increased in A549 shSnoN-expressing cells. Equal amounts of cell lysates from parental A549 or shSnoN-expressing cells were subjected to GTPase (more ...)
Cofilin is an actin-severing protein that is inactivated upon phosphorylation by Lim kinase in a pathway that proceeds downstream of RhoA activation. Phosphorylation and inactivation of cofilin result in stabilization of actin filaments and concomitant stress fiber formation (6
). Therefore, increased cofilin phosphorylation is a biochemical marker indicative of increased actin stress fiber formation. In parental lung and breast cancer cells, TGF-β stimulation resulted in phosphorylation of cofilin (Fig. ). In cells expressing shSnoN, the basal level of cofilin phosphorylation in untreated cells was heightened relative to parental cells, and TGF-β stimulation further increased the level of cofilin phosphorylation above that observed in TGF-β-treated parental cells (Fig. ). This is consistent with the observed increase in RhoA activity and stress fiber formation in these cells.
Taken together, these results indicate that the RhoA pathway appears to function downstream of SnoN to mediate its effect on EMT.
SnoN modulates multiple signaling pathways involved in the regulation of cell growth, migration, and morphology.
To further understand the signaling pathways regulated by SnoN and to confirm the biological observations outlined above, we performed microarray analysis to uncover changes in gene expression resulting from downregulation of SnoN expression. Total RNA was isolated from parental A549 cells and those expressing shRNA for SnoN and hybridized to an Affymetrix human U133 (A+B) array. Among the 44,500 total human genes on the array, the expression of 7.8% of them (3,471 genes) was altered upon downregulation of SnoN expression. Out of these 3,471 genes, 2,086 genes were upregulated and 1,348 were downregulated. An annotated list of a subset of these genes is shown in Table , and the expression of some of the relevant genes, including JunB, GADD45A, EGFR, Twist1, VEGF, PLAU, and EMP1 has been confirmed by RT-PCR (Fig. ).
Consistent with previous data suggesting that SnoN has pleiotropic effects in the regulation of tumorigenesis, SnoN-deficient cells exhibited changes in gene expression indicative of cell cycle arrest as well as EMT leading to tumor metastasis. In particular, several genes involved in negative regulation of cell cycle (p21, JunB, and cyclin G2) and apoptosis (GADD34, GADD45, and IGFBP1) were upregulated, while genes that promote cell cycle progression (cyclin E2, A2, D3, and E2F) and survival (survivin-b) were downregulated in shSnoN cells, in agreement with the reduction of tumor growth both in vitro and in vivo. Many genes involved in extracellular matrix remodeling were induced in SnoN-deficient A549 cells, including FN1, ECM2, PLAU, PAI-1, and MMP-16. In addition, expression of Twist1, an important regulator of EMT that has been shown to promote tumor cell metastasis, was elevated in SnoN-deficient cells. Thus, the ability of SnoN to inhibit cellular processes characteristic of EMT was also substantiated by microarray analysis.
As expected from its role as a negative regulator of TGF-β signaling, many SnoN-regulated genes are also TGF-β-responsive genes. These include several well-described TGF-β-inducible genes (p21
, cyclin G2
, etc.) that were upregulated in shSnoN cells as well as TGF-β-repressed genes (cyclin A2
, and TGF
) that were also downregulated in shSnoN cells (7
), suggesting that reduction of SnoN expression can enhance or repress expression of TGF-β-regulated genes, even in the absence of TGF-β stimulation (Table and Fig. and ). This is consistent with a role of SnoN in maintaining the basal states of TGF-β-responsive genes. Not surprisingly, SnoN also altered the expression of many genes not currently known to be directly involved in TGF-β signaling. These include genes involved in cell proliferation and apoptosis (IGFBP1
, etc.) and angiogenesis, EMT, and tumor metastasis/invasion (VEGF
, and autotaxin
) as well as adhesion and cell-matrix interaction (FN1
, α2 integrin
, and PLAU
). These genes contribute prominently to the complex effects of SnoN on tumor growth and progression.
Taken together, the results of the microarray analysis support cell biological and biochemical observations in SnoN-deficient cells and provide transcriptional data supporting the dual role of SnoN in tumorigenesis.