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 platelet-derived 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-kappaB, is not required for the aggressive tumorigenic phenotype in HT1080 cells. Activation of NF-kappaB 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.

FIG. 1

Western blot analysis performed on HT1080 and MCH603 cell lysates or their respective conditioned media to determine the levels of secreted PDGF-A and PDGF-B (A) or surface membrane-bound PDGFR-α and PDGFR-β (B). HT, HT1080; 603, MCH603; CM, conditioned medium.

FIG. 2

Western blot analysis of the cell lysates from HT1080/PTEN transfectants (A) and MCH603/PI3K^act transfectants (B) to determine the levels of PTEN (A), myc-tagged PI 3-kinase (B), phospho-Akt, and total Akt. Three independent HT1080/PTEN and MCH603/PI3K^act clones were analyzed. The fold level of the individual proteins (PTEN and phospho-Akt) is relative to 1.0 for HT1080 control cells. HT, HT1080; 603, MCH603.

FIG. 3

In vitro Raf, MEK, ERK, and JNK kinase assays and Elk-1 activation assays performed on HT1080/PTEN (A) and MCH603/PI3K^act (B) transfectants. For the kinase assays the fold level is relative to 1.0 for HT1080 control cells, and for the Elk-1 luciferase reporter the activities are expressed as the percent relative to 100% for HT1080. Three independent HT1080/PTEN clones and MCH603/PI3K^act clones were analyzed. HT, HT1080; 603, MCH603; V, vector only (control). The error bars indicate the standard deviations.

FIG. 4

Pull-down assays of activated Ras, Rac, and Rho. The GTP-bound forms of Ras, Rac, and Rho were pulled down with GST fusion proteins, corresponding to the Ras-binding domain of Raf-1 (Raf-1 RBD), the p21-binding domain (PBD) of human PAK-1, and the C21 binding domain of Rho, respectively, conjugated to agarose beads. The Ras-GTP, Rac-GTP, and Rho-GTP proteins bound to the beads were identified using anti-Ras (A), anti-Rac (B), and anti-Rho (C) antibodies, respectively, in a Western immunoblot. Immunoblot analysis of total cell lysates identified the levels of total protein.

FIG. 5

Western blot analysis performed on HT1080/PTEN transfectants (A) and MCH603/PI3K^act (B) transfectants to determine the levels of phospho-IkB and total IkB in these cells.

FIG. 6

In vitro luciferase reporter assays were performed to determine NF-κB activities in HT1080, HT1080/PTEN, MCH603, and MCH603/PI3K^act cells with (B) or without (A) TNF-α (TNF) treatment. Normal HDFs were used as a control for basal level activity. The NF-κB luciferase reporter activities are presented as the average fold activation. HT, HT1080; 603, MCH603.

FIG. 7

Western blot analysis performed on HT1080/PTEN transfectants (A) and 603/PI3K^act transfectants (B) to determine the level of Phospho-Bad and total Bad in these cells relative to the levels in the parental HT1080 and MCH603 cells.

FIG. 8

Actin stress fiber organization in HT1080, MCH603, HT1080/PTEN, and MCH603/PI3K^act cells. The cells were stained with fluorescein-conjugated phalloidin. Magnification, ×160.

FIG. 9

Anchorage-independent assays. Totals of 10⁴ cells (A) or 10⁶ cells (B) were plated per 60-mm petri dish in soft agar. Colonies (>0.1 mm) were counted after incubation for 3 weeks at 37°C, with periodic refeeding with fresh growth medium. The error bars indicate standard deviations.

FIG. 10

Tumorigenicity assays of HT1080, MCH603, HT1080/PTEN, and 603/PI3K^act cells. Each point is the average size of the tumor sizes of all sites inoculated (total of 6 for the parental cells and 18 for the transfectants, combining three independent clones).

FIG. 11

Summary of levels of constitutive activity of members of the PI 3-kinase, RhoA, Rac1, and Raf signaling pathways and of the tumorigenic phenotypes in parental HT1080 and MCH603 cells and their respective transfectants, HT1080/PTEN and MCH603/PI3K^act. ∗, Proteins constitutively active in HT1080 or MCH603; ↓ or ↑, decrease or increase, respectively, in the constitutive activity of individual factors tested in HT1080/PTEN and MCH603/PI3K^act relative to their respective parental cells; ↑↑, activity higher than that seen in HT1080 cells; ‡, not tested.