Truncating SOX9 Alterations Are Heterozygous Null Alleles in Genome-Stable Colorectal Cancer

Through an integrative analysis leveraging patient-derived molecular information, we recently defined the genome-stable subtype of colorectal cancer (CRC), a previously unrecognized subgroup that lacks significant aneuploidy and elevated mutational density. 1 A striking molecular feature of this new class is the presence of highly recurrent mutations in the developmental transcription factor and wingless-related integration site (WNT) pathway target SOX9. 2 However, the functional significance of these alterations in CRC remains poorly understood. Prior studies hypothesized a gain-of-function role for the mutant SOX9 based on genomic analyses of human CRC cases. 3,4 However, to date, a direct functional analysis of the role of truncated SOX9 proteins has yet to be performed. In this research letter, we annotate SOX9 mutations in CRC, describe their transcriptional and epigenomic consequences, and postulate as to why they are selected for in genome-stable CRC.

To generate lentiviruses, expression vectors were co-transfected into HEK293T cells with the lentiviral packaging constructs psPAX2 and pMD2.G (VSV-G) in a 1:1:1 ratio using X-tremeGENE 9 DNA Transfection Reagent (Roche) according to the manufacturer's instructions.Cell culture media was changed the following day and lentiviral supernatant was harvested 48 h and 72 h later and filtered through a 0.45 μm filter (Millipore).Lentiviruses were aliquoted and stored at -80 °C until use.
To perform lentiviral infection, the CRC cells were plated in a 6-cm dishes and infected with 0.5-1 mL virus in media containing 8 mg/mL polybrene overnight.

Generation of stable cell lines.
All genetically manipulated colon organoid lines were generated using the protocol described here 8 .To generate V5-tagged inducible expression of SOX9 and truncated variants, PLIX403 vectors were used and 15 μg/ml blasticidin selection was started 24 hours after infection.

RNA isolation and qPCR.
Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA) and cDNA was synthesized using the iScript TM Reverse Transcription Supermix for RT-qPCR (Bio-Rad) according to the manufacturer's instructions.Gene-specific primers for SYBR Green real-time PCR were either obtained from previously published sequences or designed by PrimerBLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and synthesized by Integrated DNA Technologies or ETON biosciences.Real-time PCR was performed and analyzed using CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA) and using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) according to the manufacturer's instructions.Relative mRNA expression was determined by normalizing to GAPDH expression, which served as an internal control.
Immunoblot and antibodies.Immunoblot analysis was performed as previously described 8 .
Briefly, cells were lysed in RIPA buffer supplemented with a protease inhibitor cocktail (Roche).
Whole cell extracts were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with indicated primary antibodies.Bound antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies and chemiluminescent HRP substrate.
The following primary antibodies were used for western blotting (all from Cell Signaling Technologies, Beverly, MA, USA, unless otherwise indicated): anti-SOX9 (#82630, 1:1,000), antiβ-Actin (A5441, 1:1,000, Sigma), and anti-V5 (R960-25, 1:2,500, Thermo Fisher) Allele frequency of the somatic mutations.To determine the allele frequency of the SOX9 somatic mutation, we queried the genomic information from colorectal adenocarcinoma patient samples included in the TCGA PanCan Atlas dataset.When the mutant allele frequency of a somatic mutation is less than or equal to 0.5, the mutation has been called as heterozygous mutation.The results are based upon data generated by TCGA and made available through cBioPortal 9 .
For the broader analysis summarized in Table 1, we performed the following steps: An initial gene list was composed using the combination of the top 20 genes mutated in CRC according to the TCGA database and most commonly mutated genes in CRC as found by Liu et al in a comprehensive genomic analysis of gastrointestinal cancers 1 .This resulted in an initial list of 50 genes, which were narrowed down to 30 genes of interest due to having known oncogenic or tumor suppressive functions.These 30 genes were queried in cBioPortal using the CRC dataset containing 526 CRC patient samples titled "Colorectal Adenocarcinoma (TCGA, PanCancer Atlas)".For each gene queried, total number of oncogenic or likely oncogenic mutations according to cBioPortal annotations were recorded in column B. The number of mutations with copy number recorded as "diploid" or "gain" were totaled in column C, and of these remaining oncogenic mutations the number with an allele fraction less than 50% was totaled and recorded in column D. The proportion heterozygous in column E was calculated by dividing column D by column B; the percentages at or below 50% have been bolded.The maximum allele fraction, percentage of total samples with a specific gene mutation, and the predicted mutation functions as annotated by cBioPortal are listed in columns F, G, and H respectively.Maximum allele fractions of 0.5 or less have been bolded.Several mutations on the original list of interest had no annotated mutations in cBioPortal, and are provided at the bottom of the table in a separate section.
Statistical Analysis and reproducibility.Data are represented as mean ± s.d unless indicated otherwise.For each experiment, either independent biological experiments or technical replicates are as noted in the figure legends.Statistical analysis was performed using Microsoft Office statistical tools or in Prism 7.0 (GraphPad).Pairwise comparisons between groups (that is, experimental versus control) were performed using an unpaired two-tailed Student's t-test or Kruskal-Wallis test as appropriate unless otherwise indicated.