TGFβ overexpression and SMAD4
mutations or deletions have been directly correlated with colon cancer metastasis. Several pathological and genetic studies suggested that chromosome 18q loss is a critical event during colorectal cancer progression and that the SMAD4
tumor suppressor is the primary target for inactivation (2
). Subsequent reports have established that allelic loss of chromosome 18q is directly correlated with liver metastasis of colorectal cancer and poor prognosis (36
). Despite the strong genetic evidence for the association between SMAD4
inactivation and advanced stage of colon cancer, the molecular basis remains elusive.
To examine if SMAD4 inactivation is a major switch that favors tumor malignancy and propensity for angiogenesis and metastasis of colon cancer, we elected to use cell line model systems to investigate both the molecular basis and cellular properties associated with SMAD4 inactivation and concurrent increase in the TGFβ levels, conditions that mimic the advanced stage colorectal tumors. Since the pairs of cell lines studied are genetically identical, except for their SMAD4 status, we reasoned that comparing the properties and gene expression patterns should help to better understand the role of SMAD4 in tumor maligancy.
Here we show that Smad4 loss enhances VEGF expression synergistically with TGFβ, whereas expression of Smad4 suppresses VEGF levels in colon cancer cells. These results are consistent with a previous report using the pancreatic cancer cell line, Hs766T, harboring homozygous deletion in both SMAD4
alleles, in which the restoration of Smad4 expression was found to suppress angiogenesis and xenograft tumor growth by inhibiting VEGF expression (38
). We also found that SMAD4
deficiency prolonged TGFβ-mediated Erk-phosphorylation and activation in HCT116 cells. The fact that Erk signaling is initially activated by TGFβ and eventually turned off at 24h in SMAD4
+/+ cells, suggests that a phosphatase may act to revert phosphorylation to the basal levels. Our results are also consistent with hyperactivation of Ras-mediated Erk signaling and progression into undifferentiated carcinoma upon inhibition of Smad4 in transformed keratinocytes (39
Interestingly, our data also showed that increased TGFβ-mediated activation of MEK-Erk and p38-MAPK pathways combined with SMAD4
loss, at least in part, mediates VEGF upregulation. This is in aggreement with studies showing that Erk kinase is required for VEGF upregulation in colon carcinoma cells upon serum starvation (32
) as well as that p38-MAPK activation by heregulin-beta-1 is required for VEGF induction in endothelial cells (33
). Our studies also found that SMAD4
inactivation in colon cancer cells enhances their migratory and invasive properties consistent with a previous report showing that restoration of Smad4 expression reversed the invasive phenotype of pancreatic cancer cells (40
Clinical studies have shown that patients retaining heterozygosity at the 18q locus benefit significantly better from treatment with 5′fluorouracil than patients with LOH at this site (34
). Moreover, chromosome 18q loss and absence of TGFβRII mutations were found to correlate with low survival rates in patients treated with adjuvant chemotherapy (41
). These clinical data are consistent with our findings using HCT116 cells harboring SMAD4
loss and intact TGFβRII status, which cooperate to induce VEGF expression. Other studies also showed a direct correlation between low levels of Smad4 in tumors and worse outcome following surgery and treatment with 5′-fluorouracil in colon cancer patients (42
Elevated glycolytic rates, even under normoxic conditions, also known as the “Warburg effect” (43
) have been correlated with the acquisition of chemoresistance in cancer cells (44
) and HIF1α is established as a major transcriptional regulator of the glucose transporter GLUT1 (46
). Interestingly, we found that SMAD4-
deficient cells exhibit increased levels of GLUT1 expression and lactate secretion as well as resistance to 5′-FU-mediated apoptosis. Since SMAD4
deficiency did not affect oxidative respiration (Figure S1
), we conclude that increased glycolysis aided by the robust glucose transport contributes to the growth advantage and enhanced survival of these cells. The fact that there was physical interaction between Smad4 and HIF1α suggests a mechanistic basis for these observations. Based on these findings we propose that Smad4 may negatively regulate HIF1α-induced GLUT1 expression and the rate of aerobic glycolysis, providing a molecular link to explain the acquisition of chemoresistance in colorectal tumors harboring chromosome 18q deficiency ().
SMAD4 inactivation promotes transition to malignancy in colon cancer
In summary, our studies provide direct evidence for a molecular basis to explain an association between a Smad4 defect and progression to malignant colon cancer (). The model systems described here may help to uncover novel biomarkers for advanced stage colon cancer to improve prognostic evaluations and identify effective targets for therapeutic intervention.