The macrophage-stimulating protein pathway promotes metastasis in a mouse model for breast cancer and predicts poor prognosis in humans

Abstract

A better understanding of tumor metastasis requires development of animal models that authentically reproduce the metastatic process. By modifying an existing mouse model of breast cancer, we discovered that macrophage-stimulating protein promoted breast tumor growth and metastasis to several organs. A special feature of our findings was the occurrence of osteolytic bone metastases, which are prominent in human breast cancer. To explore the clinical relevance of our model, we examined expression levels of three genes involved in activation of the MSP signaling pathway (MSP, MT-SP1, and MST1R) in human breast tumors. We found that overexpression of MSP, MT-SP1, and MST1R was a strong independent indicator of both metastasis and death in human breast cancer patients and significantly increased the accuracy of an existing gene expression signature for poor prognosis. These data suggest that signaling initiated by MSP is an important contributor to metastasis of breast cancer and introduce an independent biomarker for assessing the prognosis of humans with breast cancer.

Fig. 1.

Overexpression of MSP in tumors initiated by PyMT results in faster tumor growth and increased metastasis. (A) Diameter of PyMT+pMIG or PyMT+MSP tumors as a function of time after transplantation (measured upon necropsy). The data are represented as a mean ± standard deviation. (B–F) Fluorescent whole-mount images of PyMT+MSP metastases in lung, bone, and lymphatics. (E and F) Fluorescent whole-mount images of larger bone metastases in mice from which the primary tumors (PyMT+MSP) were surgically removed 6 months before analysis. (Scale bars: A, B, D–F, 1 mm; C, 100 μm.)

Fig. 2.

Tumor cells overexpressing MSP metastasized to bone, resulting in osteolytic lesions. (A) CT cross-section images of the normal left femur and the affected right femur from a mouse bearing a PyMT+MSP tumor. The images were taken at the same time, and only the right femur was affected by metastasis. (B) 3D rendering of CT data from the same mouse as in A. The arrow shows loss of bone density in the femur just above the knee. (C) Fluorescent whole-mount microscopy of the femur after the CT scan, showing a GFP+ PyMT+MSP tumor near the right knee. (D) Hematoxylin/eosin stain of a section of a normal femur. (E) Hematoxylin/eosin stain of a femur near a metastatic tumor, showing pitting of the bone beneath the periosteum (dashed line). (F) TRAP stain of the bone, showing osteoclasts in the bone pits (purple stain). The periosteum is indicated by the dashed line. (G) TRAP stain of the same bone in the region distal to the tumor. (Scale bar: C, 1 mm; D–G, 100 μm.) (H and I) In vitro bone pit assays using osteoclast-like cells derived from mouse bone marrow and cocultured with PyMT+pMIG primary tumor cells (H) or PyMT+MSP bone metastasis tumor cells (I). The bone matrix stains brown, and the pits induced by osteoclasts appear as white patches. (Scale bars, 100 μm.)

Fig. 3.

Overexpression of the MSP/MST1R pathway in human breast cancer is associated with metastasis and death. (A) Kaplan–Meier analysis showing development of metastases over time for two groups of patients: those whose tumors expressed MSP/MST1R/MT-SP1 (according to MSP oligonucleotide 2) at levels above the mean (red line), and those whose tumors did not express all three genes above the mean (blue line). (B) Kaplan–Meier analysis showing time to death for the same groups of patients as in A. P values were calculated by using the log-rank test.