Dissection of Ras-dependent signaling pathways controlling aggressive tumor growth of human fibrosarcoma cells: evidence for a potential novel pathway

Abstract

Activation of multiple signaling pathways is required to trigger the full spectrum of in vitro and in vivo phenotypic traits associated with neoplastic transformation by oncogenic Ras. To determine which of these pathways are important for N-ras tumorigenesis in human cancer cells and also to investigate the possibility of cross talk among the pathways, we have utilized a human fibrosarcoma cell line (HT1080), which contains an endogenous mutated allele of the N-ras gene, and its derivative (MCH603c8), which lacks the mutant N-ras allele. We have stably transfected MCH603c8 and HT1080 cells with activating or dominant-negative mutant cDNAs, respectively, of various components of the Raf, Rac, and RhoA pathways. In previous studies with these cell lines we showed that loss of mutant Ras function results in dramatic changes in the in vitro phenotypic traits and conversion to a weakly tumorigenic phenotype in vivo. We report here that only overexpression of activated MEK contributed significantly to the conversion of MCH603c8 cells to an aggressive tumorigenic phenotype. Furthermore, we have demonstrated that blocking the constitutive activation of the Raf-MEK, Rac, or RhoA pathway alone is not sufficient to block the aggressive tumorigenic phenotype of HT1080, despite affecting a number of in vitro-transformed phenotypic traits. We have also demonstrated the possibility of bidirectional cross talk between the Raf-MEK-ERK pathway and the Rac-JNK or RhoA pathway. Finally, overexpression of activated MEK in MCH603c8 cells appears to result in the activation of an as-yet-unidentified target(s) that is critical for the aggressive tumorigenic phenotype.

FIG. 1

Pull-down assays of activated Ras, Rac, and RhoA. The GTP-bound forms of Ras, Rac, and RhoA were pulled down with glutathione S-transferase fusion proteins, corresponding to the Ras binding domain of Raf-1, PBD of human PAK-1, and C21 binding domain of RhoA, respectively, conjugated to agarose beads. The Ras-GTP, Rac-GTP, and RhoA-GTP proteins bound to the beads were identified using anti-Ras (A) anti-Rac (B), and anti-RhoA (C) antibodies, respectively, in a Western immunoblot. Immunoblot analysis of total cell lysates identified the levels of total protein. HT, HT1080 cells; 603, MCH603c8 cells; V, vector-only control.

FIG. 2

In vitro Raf, MEK, ERK, and JNK kinase assays and Elk-1 activation assays performed on HT1080 (HT)-Raf DN transfectants (A) and MCH603c8 (603)-Raf^act transfectants (B). For the kinase assays, the fold level is relative to 1.0 for HT1080 control cells, and the Elk-1 luciferase reporter activities are expressed as percents relative to 100% for HT1080. Three independent Raf DN clones and Raf^act clones were analyzed. V, vector-only control. The error bars indicate standard deviations.

FIG. 3

In vitro Raf, MEK, ERK, and JNK kinase assays and Elk-1 activation assays performed on HT1080 (HT)-MEK DN transfectants (A) and MCH603c8 (603)-MEK^act transfectants (B). Fold levels were calculated as for Fig. 2. Three independent MEK DN clones and MEK^act clones were analyzed. V, vector-only control. The error bars indicate standard deviations.

FIG. 4

In vitro Raf, MEK, ERK, and JNK kinase assays and Elk-1 activation assays performed on HT1080(HT)-Rac DN transfectants (A) and MCH603c8 (603)-Rac^act transfectants (B). Fold levels were calculated as for Fig. 2. Three independent Rac DN clones and Rac^act clones were analyzed. V, vector-only control. The error bars indicate standard deviations.

FIG. 5

Actin stress fiber organization in parental HT1080 (HT) and MCH603c8 (603) cells and their respective DN and constitutively active mutant transfectants. The cells were stained with fluorescein-conjugated phalloidin. The arrows indicate the enhancement of adhesion plaques. Magnification, ×200

FIG. 6

In vitro growth kinetics of parental HT1080 (HT) and MCH603c8 (603) cells and their respective DN and constitutively active mutant transfectants. The error bars indicate standard deviation.

FIG. 7

Anchorage-independent assays. A total of 10⁴ (A and C) and 10⁶ (B and D) cells were plated per 60-mm-diameter petri dish in soft agar. Colonies (>0.1-mm diameter) were counted after incubation for 3 weeks at 37°C, with periodic refeeding with fresh growth medium. HT, HT1080 cells; 603, MCH603c8 cells; V, vector-only control. The error bars indicate standard deviations.

FIG. 8

Tumorigenicity assays of HT1080 (HT) and HT1080-DN transfectants (A) and MCH603c8 (603) and MCH603c8-activating mutant transfectants (B). Each point is the average of the tumor sizes of all sites inoculated (a total of 6 for parental cells and 18 for the transfectants, combining three independent clones). cumm, cubic millimeters.

FIG. 9

Schematic diagrams of the downregulated and activated status of the downstream members of the Ras-mediated signaling pathways in the transfectants compared to those of the parental HT1080 and MCH603c8 cells. The down and up arrows indicate downregulation and activation of kinase activity, respectively, of those proteins that has occurred as a consequence of expression of transfected DN (HT1080-Raf DN, HT1080-MEK DN, and HT1080-Rac DN) and activated (MCH603c8-Raf^act, MCH603c8-MEK^act, and MCH603c8-Rac^act) mutants, respectively. The horizontal and diagonal arrows indicate probable cross talk between the Raf and Rac1 pathways. The positive or negative cross talk may be direct or indirect. Also, the specific level at which the cross talk takes place is not clear in every case.