G van der Pluijm

Smad2 and Smad3 have opposing roles in breast cancer bone metastasis by differentially affecting tumor angiogenesis

Transforming growth factor (TGF)-β can suppress and promote breast cancer progression. How TGF-β elicits these dichotomous functions and which roles the principle intracellular effector proteins Smad2 and Smad3 have therein, is unclear. Here, we investigated the specific functions of Smad2 and Smad3 in TGF-β-induced responses in breast cancer cells in vitro and in a mouse model for breast cancer metastasis. We stably knocked down Smad2 or Smad3 expression in MDA-MB-231 breast cancer cells. The TGF-β-induced Smad3-mediated transcriptional response was mitigated and enhanced by Smad3 and Smad2 knockdown, respectively. This response was also seen for TGF-β-induced vascular endothelial growth factor (VEGF) expression. TGF-β induction of key target genes involved in bone metastasis, were found to be dependent on Smad3 but not Smad2. Strikingly, whereas knockdown of Smad3 in MDA-MB-231 resulted in prolonged latency and delayed growth of bone metastasis, Smad2 knockdown resulted in a more aggressive phenotype compared with control MDA-MB-231 cells. Consistent with differential effects of Smad knockdown on TGF-β-induced VEGF expression, these opposing effects of Smad2 versus Smad3 could be directly correlated with divergence in the regulation of tumor angiogenesis in vivo. Thus, Smad2 and Smad3 differentially affect breast cancer bone metastasis formation in vivo.

CRIPTO and its signaling partner GRP78 drive the metastatic phenotype in human osteotropic prostate cancer.

CRIPTO (CR-1, TDGF1) is a cell surface/secreted oncoprotein actively involved in development and cancer. Here, we report that high expression of CRIPTO correlates with poor survival in stratified risk groups of prostate cancer (PCa) patients. CRIPTO and its signaling partner glucose-regulated protein 78 (GRP78) are highly expressed in PCa metastases and display higher levels in the metastatic ALDHhigh sub-population of PC-3M-Pro4Luc2 PCa cells compared with non-metastatic ALDHlow. Coculture of the osteotropic PC-3M-Pro4Luc2 PCa cells with differentiated primary human osteoblasts induced CRIPTO and GRP78 expression in cancer cells and increases the size of the ALDHhigh sub-population. Additionally, CRIPTO or GRP78 knockdown decreases proliferation, migration, clonogenicity and the size of the metastasis-initiating ALDHhigh sub-population. CRIPTO knockdown reduces the invasion of PC-3M-Pro4Luc2 cells in zebrafish and inhibits bone metastasis in a preclinical mouse model. These results highlight a functional role for CRIPTO and GRP78 in PCa metastasis and suggest that targeting CRIPTO/GRP78 signaling may have significant therapeutic potential.

Noninvasive real-time in vivo bioluminescent imaging of gene expression and of tumor progression and metastasis.

It is clear from the work presented in this chapter and from work by others that BLI is perfectly suited to monitor gene expression in transgenic reporter mice and to detect and follow small numbers of cells noninvasively. As we have shown, it also enables the quantification of tumor cells within internal organs in animal models of cancer. BLI is a powerful tool in functional genomics of cancer development, progression, and metastasis and will allow us to identify in vivo molecular targets of cancer and their metastasis. The application of BLI in combination with new animal models for cancer will allow us to study very rapidly and conveniently the efficacy of new therapeutic approaches such as gene therapy stem cell therapy and antiangiogenic therapy, and when successful can be a first step towards clinical application. Furthermore, the development of new smart luciferase-based reporter constructs as well as new possibilities to create transgenic animals containing these reporter constructs will make noninvasive in vivo BLI also a powerful new tool in other small animal models of human biology and disease.

Monitoring metastatic behavior of human breast cancer cells in mice with species-specific PCR and bioluminescent reporter imaging

Monitoring metastatic behavior of human breast cancer cells in mice with species-specific PCR and bioluminescent reporter imaging