Targeting Cancer Progression Genes Upregulated in CREB3L1‐Deficient Breast Cancer Cells
Authors: Smith Shari , Goubran Farah , Mellor Paul , Anderson Deborah 
Published: November 2023

Background

Our laboratory discovered that CREB3L1 (cAMP responsive element binding protein 3‐like protein), a stress‐activated transcription factor, acts as a metastasis suppressor in breast cancer. CREB3L1 is expressed ubiquitously in non‐cancerous human breast cells and restricts expression of genes that promote cell growth, angiogenesis, and migration. Loss of CREB3L1 expression, which is a frequent event in high grade metastatic human breast tumors, can result in enhanced metastatic properties. To establish the mechanisms by which CREB3L1 exerts its tumor suppressive effects, a gene expression microarray analysis was performed to determine which genes are upregulated in CREB3L1‐deficient breast cancer cells. Six hundred and eighty genes were statistically significantly upregulated at least 2‐fold in all 4 of the CREB3L1‐deficient cell lines assessed. To focus our analysis, 4 genes were identified as potential targets for this study, given that they have previously been shown to have a role in cancer progression.

Objective

Gene expression of the 4 test genes (Genes 1, 2, 3, 4) are upregulated in CREB3L1‐deficient human breast cancer cells. Cells that had knockdown of each one of these 4 targets were extensively characterized to determine the role of each target in breast cancer tumorigenesis and metastasis.

Hypothesis

Inhibiting one or more of the cancer progression genes upregulated in CREB3L1‐deficient breast cancer cell lines will repress their tumorigenic and/or migratory properties.

Methods

Knockdowns were generated for each of the 4 genes in a CREB3L1‐deficient breast cancer cell line (HCC1806) and matched CREB3L1‐expressing cells (HCC1806 + HA‐CREB3L1), as well as a non‐tumorigenic control breast cell line (MCF10A). To knockdown expression of each of the 4 targets, shRNA directed at the gene of interest was transduced via a lentiviral system into the cells. Cell‐based assays were performed to characterize the properties of these cell lines, which included the Boyden chamber assay to measure migration, soft agar assay to assess anchorage‐independent growth, and the MTT assay to evaluate cell proliferation.

Results

Successful knockdown at both the mRNA and protein level for each target gene was validated using quantitative real time PCR and immunoblotting. Results from the MTT assay verified that knockdown of each of the 4 gene targets did not affect cell survival in MCF10A cells, confirming that these test genes are not essential for survival in non‐cancerous breast cells (p > 0.05). Similarly, knockdown of each of the test genes did not affect cell proliferation in HCC1806 (± CREB3L1) cells, except for a significant decrease in proliferation in HCC1806 CREB3L1‐deficient cells with Gene 1 knockdown (p =0.02). Preliminary results indicate that knockdown of Genes 1, 2, and 4 in HCC1806 CREB3L1‐deficient cells reduces cancer cell properties, including cell migration and anchorage‐independent growth (p < 0.05).

Conclusion

Promising candidates will be tested in follow‐up mouse xenograft studies. Positive results in these pre‐clinical models would provide a strong rationale for the development and testing of inhibitors to one or more of these potential targets as a new therapeutic for the 30% of breast cancers that are CREB3L1‐deficient.

Support or Funding Information

This research received funding from Saskatchewan Health Research Foundation and the Canadian Institutes of Health Research.