Short-Form Ron Promotes Spontaneous Breast Cancer Metastasis through Interaction with Phosphoinositide 3-Kinase

Abstract

Receptor tyrosine kinases (RTKs) have been the subject of intense investigation due to their widespread deregulation in cancer and the prospect of developing targeted therapeutics against these proteins. The Ron RTK has been implicated in tumor aggressiveness and is a developing target for therapy, but its function in tumor progression and metastasis is not fully understood. We examined Ron activity in human breast cancers and found striking predominance of an activated Ron isoform known as short-form Ron (sfRon), whose function in breast tumors has not been explored. We found that sfRon plays a significant role in aggressiveness of breast cancer in vitro and in vivo. sfRon expression was sufficient to convert slow-growing, nonmetastatic tumors into rapidly growing tumors that spontaneously metastasized to liver and bones. Mechanistic studies revealed that sfRon promotes epithelial-mesenchymal transition, invasion, tumor growth, and metastasis through interaction with p85, the regulatory subunit of phosphoinositide 3-kinase (PI3K). Inhibition of PI3K activity, or introduction of a single mutation in the p85 docking site on sfRon, completely eliminated the ability of sfRon to promote tumor growth, invasion, and metastasis. These findings reveal sfRon as an important new player in breast cancer and validate Ron and PI3K as therapeutic targets in this disease.

Keywords: PI3K; Ron; breast cancer; metastasis; short-form Ron.

Figure 1.

sfRon is the major active Ron isoform in tumors from breast cancer patients. (A) Representative Western blot of breast tumor lysates from 6 different patients using antibodies specific for the C-terminus of Ron (C-20; upper blot) or those specific for active, phosphorylated Ron (pRon Y1238/1239; lower blot). (B) Representative Western blot of breast tissue lysates from 10 different patients following reduction mammoplasty using antibodies specific for the C-terminus of Ron (C-20; upper blot) or those specific for active, phosphorylated Ron (pRon Y1238/1239; lower blot). Tumor 4 is the same sample as that shown in A. The line on the top gel denotes separation of 2 different film exposures from the same blot. The proform of Ron (proRon), Ron β chain (Ron), and sfRon (or phosphorylated sfRon [p-sfRon]) are indicated. The putative ubiquitylated sfRon (sfRon-HMW) is also noted.

Figure 2.

sfRon confers EMT and invasive capability in breast cancer cells. (A) Left panel: proliferation of MCF7 and MCF7-sfRon cells based on mitochondrial dehydrogenase activity, read as absorbance over time. Right panel: survival of MCF7 and MCF7-sfRon cells in serum-free conditions based on mitochondrial dehydrogenase activity. (B) Phase-contrast micrograph illustrating altered morphology of MCF7 cells expressing sfRon (right panel) compared to those expressing full-length Ron (left panel). Photographs were taken at the same magnification; scale bars represent 100 µm. (C) Western blot with antibodies specific for E-cadherin, N-cadherin, and β-actin showed that sfRon decreased expression of E-cadherin and increased expression of N-cadherin. (D) sfRon induced invasion, as quantified by determining the number of cells that passed through Matrigel-coated Boyden chambers. No significant change was observed in MCF7-Ron cells. White bars: random invasion toward serum-free medium; gray bars: invasion toward medium containing 10% serum. Error bars reflect standard deviation from at least 3 experimental replicates.

Figure 3.

PI3K signaling is activated by sfRon and is required for the invasive activity of sfRon. (A) Western blot on whole-cell lysates from parental MCF7 cells or those expressing Ron or sfRon, using antibodies specific for Akt or Erk (phosphorylated or total). (B) Addition of PI3K inhibitors Ly294002 (40 uM) or wortmannin (1, 5, or 10 nM) blocked sfRon-induced invasion. Error bars reflect standard deviation from at least 3 experimental replicates. (C) Western analysis of N-cadherin on wortmannin-treated MCF7-sfRon cells. The results revealed that blocking PI3K signaling could not reverse sfRon-induced EMT.

Figure 4.

PI3K activation through interaction with sfRon is required for EMT and invasion. (A) Diagram showing mutation of the sfRon docking site (2G), designed to disrupt PI3K binding and facilitate Grb2 binding. (B) Western blot demonstrating that the sfRon2G mutation did not change sfRon and phosphorylated sfRon (p-sfRon, detected with anti-pY1238/39 Ron) expression in MCF7 cells (top 2 panels). Lower panels: Western blot for sfRon following GST pull-down assays using the SH2 domain of Grb2 (Grb2SH2) or the SH2 domain of the p85 subunit of PI3K (p85SH2). The amount of GST protein used in the assays is shown in the lowest panel. (C) Western blot showing that sfRon2G promotes MAPK signaling (shown by p-Erk) instead of PI3K signaling (shown by p-Akt). (D) The sfRon2G mutant failed to induce EMT in MCF7 cells, as assessed by Western blotting for E-cadherin and N-cadherin proteins. (E) The sfRon2G mutant was defective for invasion ability, as measured by quantification of cells that crossed the Matrigel-coated membrane in a Boyden chamber assay.

Figure 5.

sfRon expression increases tumor growth through activation of PI3K. Orthotopic tumor growth rate for MCF7, MCF7-Ron, MCF7-sfRon, and MCF-sfRon2G cells upon orthotopic injection into mammary fat pads of NOD/SCID mice (n = 5-7 per group; error bars reflect standard deviations).