Role of Phosphoinositide 3-Kinase in the Aggressive Tumor Growth of HT1080 Human Fibrosarcoma Cells

Abstract

We have developed a model system of human fibrosarcoma cell lines that do or do not possess and express an oncogenic mutant allele of N-ras. HT1080 cells contain an endogenous mutant allele of N-ras, whereas the derivative MCH603 cell line contains only wild-type N-ras. In an earlier study (S. Gupta et al., Mol. Cell. Biol. 20:9294-9306, 2000), we had shown that HT1080 cells produce rapidly growing, aggressive tumors in athymic nude mice, whereas MCH603 cells produced more slowly growing tumors and was termed weakly tumorigenic. An extensive analysis of the Ras signaling pathways (Raf, Rac1, and RhoA) provided evidence for a potential novel pathway that was critical for the aggressive tumorigenic phenotype and could be activated by elevated levels of constitutively active MEK. In this study we examined the role of phosphoinositide 3-kinase (PI 3-kinase) in the regulation of the transformed and aggressive tumorigenic phenotypes expressed in HT1080 cells. Both HT1080 (mutant N-ras) and MCH603 (wild-type N-ras) have similar levels of constitutively active Akt, a downstream target of activated PI 3-kinase. We find that both cell lines constitutively express plateletderived growth factor (PDGF) and PDGF receptors. Transfection with tumor suppressor PTEN cDNA into HT1080 and constitutively active PI 3-kinase-CAAX cDNA into MCH603 cells, respectively, resulted in several interesting and novel observations. Activation of the PI 3-kinase/Akt pathway, including NF-B, is not required for the aggressive tumorigenic phenotype in HT1080 cells. Activation of NF-B is complex: in MCH603 cells it is mediated by Akt, whereas in HT1080 cells activation also involves other pathway(s) that are activated by mutant Ras. A threshold level of activation of PI 3-kinase is required in MCH603 cells before stimulatory cross talk to the RhoA, Rac1, and Raf pathways occurs, without a corresponding activation of Ras. The increased levels of activation seen were similar to those observed in HT1080 cells, except for Raf and MEK, which were more active than HT1080 levels. This cross talk results in conversion to the aggressive tumorigenic phenotype. This latter observation is consistent with our previous observation that overstimulation of the activity of endogenous members of Ras signaling pathways, activated MEK in particular, is a prerequisite for aggressive tumorigenic growth.

3-Methyladenine suppresses cell migration and invasion of HT1080 fibrosarcoma cells through inhibiting phosphoinositide 3-kinases independently of autophagy inhibition

“…HT1080 cells have been shown to exhibit both mesenchymal and amoeboid types of cell migration that are dependent on Rac and Rho signaling, respectively (22). PI3K-C2ß is probably stimulated in HT1080 cells because the cells constitutively express PDGF and PDGF receptors (29). These facts together with the present observations suggested that 3-MA might inhibit the activation of Rac in HT1080 cells.…”

“…However, we could not detect any change in the activation state of Rac in 3-MA-treated cells, indicating that the level of Rac constitutive activity is PI3K-independent. Presumably, the level is mediated by the mutant N-Ras protein expressed in the cells, as suggested previously (29).…”

HIF-1α mRNA and protein upregulation involves Rho GTPase expression during hypoxia in renal cell carcinoma

“…This effect appears to involve PI-3K activity, which is known to be downstream of Ras, since its inhibition by wortmannin prevents Rac1 and HIF-1 transcriptional induction in response to hypoxia (Hirota and Semenza, 2001;Jiang et al, 2001). Other studies have also demonstrated that PI-3K may activate Rho proteins such as RhoA and Rac1 in human fibrosarcoma cells (Gupta et al, 2001). Together, these data suggest that PI-3K activity is an upstream event mediating Rho GTPases activation by ROS during hypoxia in RCC.…”

Platelet-derived growth factor-C functions as a growth factor in mouse embryonic stem cells and human fibrosarcoma cells

“…1a). In addition, as reported previously [28], we confirmed that HT1080 cells express both PDGF-Rα and β (Fig. 1b).…”

“…In HT1080 fibrosarcoma cells, a decrease in Ras signaling due to the loss of an activated mutation in the N-ras gene reduced their anchorage-independent growth, and this reduction was restored through the ectopic expression of activated MEK [37]. By contrast, the overexpression of PTEN, an inhibitor of the PI3K/Akt pathway, has shown no effect on the cells' ability to form colonies in soft agar [28]. These results suggest that PDGF-C activates the Ras/Erk pathway, but not the PI3K/Akt pathway, to promote HT1080 cell growth.…”