The unfolded protein response (UPR) is activated in response to hypoxia-induced stress such as in the tumor microenvironment. This study examined the role of CREB3L1 (cyclic AMP [cAMP]-responsive element-binding protein 3-like protein 1), a member of the UPR, in breast cancer development and metastasis. Initial experiments identified the loss of CREB3L1 expression in metastatic breast cancer cell lines compared to low-metastasis or nonmetastatic cell lines. When metastatic cells were transfected with CREB3L1, they demonstrated reduced invasion and migration in vitro, as well as a significantly decreased ability to survive under nonadherent or hypoxic conditions. Interestingly, in an in vivo rat mammary tumor model, not only did CREB3L1-expressing cells fail to form metastases compared to CREB3L1 null cells but regression of the primary tumors was seen in 70% of the animals as a result of impaired angiogenesis. Microarray and chromatin immunoprecipitation with microarray technology (ChIP on Chip) analyses identified changes in the expression of many genes involved in cancer development and metastasis, including a decrease in those involved in angiogenesis. These data suggest that CREB3L1 plays an important role in suppressing tumorigenesis and that loss of expression is required for the development of a metastatic phenotype.

The unfolded protein response (UPR) is required for proper protein folding in the endoplasmic reticulum under conditions of stress such as those encountered in the tumor microenvironment. Recently, a number of studies have suggested a role for the unfolded protein response in the development of cancer, more specifically in regulating the balance between cell death, dormancy and the growth of cancer cells in the tumor microenvironment. This study examined the role of the transcription factor CREB3L1, a member of the UPR machinery, in breast cancer development and metastasis. Northern blotting analysis identified a loss of CREB3L1 expression in metastatic breast cancer cell lines compared to low- or non-metastatic cell lines, which was subsequently shown, through bisulphate sequencing, to be the result of enhanced promoter methylation. When metastatic cells were transfected with CREB3L1 they demonstrated a significant decrease in survival in hypoxic and non-adherent growth conditions, and reduced in vitro invasion and migration in chemotaxis assays. Interestingly, in an in vivo rat mammary tumor model where rats were injected with cells in the hind footpad, the CREB3L1 expressing rat cells not only failed to form metastases but tumor regression was seen in 70% of the animals. In contrast, metastases were seen in the popliteal lymph node of 90% of rats injected with wild-type and empty vector-transfected cells. Microarray and ChIP on Chip analyses identified changes in the expression of many genes involved in cancer development and metastasis, including a decrease in those involved in angiogenesis when CREB3L1 was expressed. We also observed a significant decrease in the number of migrating endothelial cells in scratch and chemotaxis assays in media conditioned by CREB3L1 expressing cells, compared to conditioned media from untransfected cells. To determine if these findings translated to human breast cancer, real-time PCR analysis of CREB3L1 expression in human breast cancer tissues of differing grade (n=30) was performed. Expression of CREB3L1 was elevated in low and medium grade tissue but substantially reduced in high grade tissue, compared to normal. The high grade breast cancer tissues demonstrated a 6.6 fold decrease in the expression of CREB3L1 compared to the low grade tissues (p<0.001), with about a 2 fold decrease compared to normal tissue. In conclusion, CREB3L1 expression is elevated in low and medium grade breast cancer tissue, perhaps as the cancer cells respond to various stresses. However, loss of CREB3L1 expression is required for the long-term survival of breast cancer cells in the tumor microenvironment and the progression to a highly metastatic phenotype. CREB3L1 expression may be a useful marker in predicting tumor grade and patient prognosis, in addition to providing a therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1450. doi:10.1158/1538-7445.AM2011-1450