Gene array analysis of clear cell renal cell carcinoma cell lines

treated with A939572.

Amanda Vargas
One of the most prevalent types of kidney cancer in adults is clear cell renal cell carcinoma
(ccRCC). The tumour arises from the cells of the proximal renal tubular epithelium. It is
considered an adenocarcinoma There are two subtypes: sporadic (that is, non-hereditary) and
hereditary. Both such subtypes are associated with mutations in the short-arm of chromosome
3, with the implicated genes being either tumour suppressor genes (VHL and TSC) or
oncogenes Unfortunately, there are limited targeted therapies available to treat ccRCC in
patients. Stearoyl-CoA desaturase 1 (SCD1) is upregulated in ccRCC tumors and has been
identified as a molecular target for ccRCC treatment. A compound, A939572, inhibits SCD1
resulting in decreased cell proliferation and increased cell death in ccRCC tumors. A 2013 study
by von Roemeling et al. aimed to identify other potential biomarkers involved in the mechanisms
of decreased cell growth and apoptosis during SCD1 inhibition by A939572 that could be used
in response to anti-SCD1 therapy.
The microarray data was generated by von Roemeling et al. The data includes four clear cell
renal cell carcinoma cell lines: ACHN ccRCC, A498 ccRCC, CAKI1 ccRCC, and CAKI2 ccRCC
each treated with DMSO and A939572 (a stearoyl-CoA desaturase 1 (SCD1) inhibitor) yielding
eight total samples for gene expression analysis. Gene expression analysis was conducted
using Affymetrix Human Genome U133 Plus 2.0 Array chip and loaded into R for analysis using
the Affy package.
The scatterplox matrices of the log2 expression data for all 8 samples was generated. The
expression values were normalized and the data was combined into probeset summaries using
RMA. Limma was used to calculate the p-values using a paired t-test and the fold change for all
probesets. Each cell line was treated as a biological replicate so we created a block for cell line
in order for limma to do a paired t-test. A histogram of the correlations was generated for DMSO
vs A939572 and the consensus correlation was checked to see if it looked reasonable given the
histogram. The contrast matrix for DMSO vs A939572 was generated. A paired t-test was
performed for all probesets using Limma. A histogram of the pvalues was generated for the
DMSO vs A939572 contrast. The topTable command was used to generate a list of the most
significant probesets. These genes had the largest fold changes indicating biological
significance. The identified probesets were then matched to the appropriate corresponding
gene(s)

For this study there were no technical replicates and just four biological replicates (the cell lines)
but for future studies it might be beneficial to look at a single cell line generated from multiple
subjects or plated out as technical replicates to see if there is still significant differential gene
expression for ATF3 and the other ER stress response genes. The statistical analysis is
replicable. Though a time course study would also be interesting to see how the ER stress
response genes react to prolonged SCD1 inhibition. Unfortunately, due to the small sample set
and lack of replicates, not much statistical information could be derived from this microarray data
though plenty of biological significance was obtained.