Shp2 suppresses PyMT-induced transformation in mouse fibroblasts by inhibiting Stat3 activity

Abstract

We have examined the effect of expression of the protein tyrosine phosphatase Shp2 on transformation by the mouse polyoma virus middle T antigen (PyMT). Gain-of-function mutations in Shp2 indicate that it may serve as an oncogene in several types of human leukemia. Paradoxically, however, some catalytically dominant-negative mutations of Shp2 have also been identified in leukemia and neuroblastomas. In this study, we show that Shp2 suppresses transformation induced by PyMT, the major polyoma viral oncoprotein known to act through binding and activation of pp60(c-src). Over-expression of a catalytically inactive Shp2 mutant in NIH3T3 cells significantly enhanced PyMT-induced transformation. Conversely, re-introduction of Shp2 into Shp2-deficient cells strongly inhibited PyMT-induced transformation and tumorigenesis. Short hairpin RNA (shRNA)-mediated Shp2 knockdown potentiated PyMT-induced transformation. Finally, we present evidence that the transformation-suppressive effects of Shp2 are mediated at least partially through the inhibition of signal transducers and activators of transcription 3.

Figure 1

Overexpression of a dominant negative Shp2 mutant enhanced PyMT-induced transformation. (A) PyMT induced transformation in 3T3 cells transduced with empty vector (3T3/Vector) or PyMT (3T3.PyMT) as indicated. Top panel: Western blot of PyMT with tubulin as a loading control. Middle panel: Focus formation assay of cells as indicated. Bottom panel: Quantitative results of focus formation using NIH Image J software. Values represent relative color density. Data shown are representative of three independent experiments. (B) Overexpression of dominant-negative Shp2 mutant increased transformation in PyMT-transformed 3T3 cells. Top panel: Western blot of Shp2. PyMT-3T3 cells transduced with empty vector (Vector), wild type Shp2 (Shp2 WT), or Shp2 C/S mutant (Shp2 C/S). Middle panel: Focus formation assays of the cells as indicated. Bottom panel: Quantitative result of focus formation assays.

Figure 2

Restoring Shp2 expression in Shp2-deficient cells inhibited PyMT-induced transformation in vitro and in vivo. Shp2-rescued B1R cells were established by restoring Shp2 expression in Shp2-deficient B1cells. (A) PyMT induced transformation in B1 or B1R cells transduced with empty vector (B1/Vector or B1R/Vector) or PyMT (B1/PyMT or B1R/PyMT) as indicated. Top panel: Western blot of PyMT or Shp2. Middle panel: Focus formation assays of cells as indicated. Bottom panel: Quantitative results of focus formation. (B) The expression of exogenous Shp2 in B1R cell is comparable to that of endogenous level in wild type fibroblast cells. Western blot analysis was performed using the antibodies as indicated in A. (C) PyMT-induced transformation in vivo is inhibited in Shp2-rescued B1R cells. Top panel: Tumor formation in the mice subcutaneously inoculated with cells as indicated. Pictures were taken three weeks after cell inoculation. Please note that a small pulp corresponding to B1/Vector is granulation tissue that was identified by histological staining (data not shown). Middle panel: Quantitative result of the tumor size (n= ±SD). Bottom panel: Histological H&E staining of the dissected tumors.

Figure 3

Shp2 knockdown enhanced PyMT-induced transformation. PyMT-transformed 3T3 cells were transduced with scrambled shRNA or Shp2 shRNA for 6 and 10 days. (A) The knockdown efficiency of Shp2 shRNA. Left panel: Western blot of Shp2 and ERK2 as a loading control. Right panel: Quantitative result of the Western blots using NIH Image J software. The value for each band represents relative band density normalized to the loading control. Data shown are representative of three independent experiments. (B) Right panel: Focus formation assays. Left panel: Quantitative result.

Figure 4

Stat3 partially mediates the effect of Shp2 on PyMT-induced transformation. (A) The phosphorylation level of Stat3 was increased in Shp2-deficient cells. The empty vector- or PyMT-transduced B1 or B1R cells were serum-starved overnight and then stimulated with EGF (100 ng/ml), followed by Western blotting with the antibodies as indicated. Arrow heads indicate quantitative results of phospho-Stat3. (B) Shp2 knockdown enhanced the phosphorylation of Stat3 in PyMT-expressing 3T3 cells. Western blots were performed on 3T3 cells transduced with Shp2 shRNA 6 and 10 days after infection. (C, D) Stat3 knockdown suppressed PyMT-induced transformation. PyMT-transduced B1 or B1R cells were transduced with Scrambled or Stat3 shRNA lentiviruses, followed by Western blotting (C) or focus formation assay (D). The left panel in (D) is a quantitative result of the focus formation.