Suprabasin Is Hypomethylated and Associated with Metastasis in Salivary Adenoid Cystic Carcinoma

<div><h3>Background</h3><p>Salivary gland adenoid cystic carcinoma (ACC) is a rare cancer, accounting for only 1% of all head and neck malignancies. ACC is well known for perineural invasion and distant metastasis, but its underlying molecular mechanisms of carcinogenesis are still unclear.</p> <h3>Principal Findings</h3><p>Here, we show that a novel oncogenic candidate, suprabasin (SBSN), plays important roles in maintaining the anchorage-independent and anchorage-dependent cell proliferation in ACC by using SBSN shRNA stably transfected ACC cell line clones. SBSN is also important in maintaining the invasive/metastatic capability in ACC by Matrigel invasion assay. More interestingly, SBSN transcription is significantly upregulated by DNA demethylation induced by 5-aza-2′-deoxycytidine plus trichostatin A treatment and the DNA methylation levels of the SBSN CpG island located in the second intron were validated to be significantly hypomethylated in primary ACC samples versus normal salivary gland tissues.</p> <h3>Conclusions/Significance</h3><p>Taken together, these results support SBSN as novel oncogene candidate in ACC, and the methylation changes could be a promising biomarker for ACC.</p> </div

<i>SBSN</i> is important in maintaining anchorage-independent and anchorage-dependent growth in SACC83.

<p>Scramble, shRNA 1, shRNA 2, and shRNA 3 indicate the control and three types of <i>SBSN</i> shRNAs used to establish stable clones in SACC83. A, Representative photographs of anchorage-independent growth by soft agar assay. The size of colonies indicates the focus formation ability of each type of stable clone. B, The number of colonies counted in each type of stable clone. C, Anchorage-dependent cell proliferation assay. CCK-8 absorbance indicates the amount of cells at time-points 0, 24, and 48 hours. Scramble clones grew faster than <i>SBSN</i> shRNA stable clones at 24 and 48 hours, p<0.01. Statistical comparisons were performed with Student’s t-test. Error bars indicate the standard deviation of triplicate assays. D. <i>SBSN</i> mRNA levels in different stable clones were determined by qRT-PCR. This confirmed that <i>SBSN</i> was silenced by ∼50% in shRNAs clones compared to scramble clones.</p

<i>SBSN</i> is hypomethylated in primary ACC samples, and its expression is induced by CpG island demethylation in SACC83.

<p>A. We first confirmed 5-aza-dC/TSA-induced expression of <i>SBSN</i> in SACC83 mRNA levels by qRT-PCR. B. Graph of actual data using TaqMan qRT-PCR analysis. X-axis, amplification cycle numbers; y-axis, ΔRn values used to plot signal attributable to the 5′ nuclease reaction, which reflects the quantity of amplicon. <i>SBSN</i> was amplified between 25–30 cycles in 5-aza-dC/TSA treatment, whereas it was amplified between 30–35 cycles in mock treatment. 5-aza-dC/TSA- or mock-treated samples were loaded in the same amount as indicated by <i>GAPDH</i>. C. qMSP was conducted in a paraffin-embedded ACC cohort, which consisted of 62 ACC samples and 25 normal salivary gland tissues. Significant hypomethylation in <i>SBSN</i> was shown in ACC versus normal salivary gland tissue (p<0.0001, Student’s t-test). <i>SBSN</i> methylation scores were normalized by β-actin. Error bars indicate the standard deviation.</p

Clinical and pathologic characteristics of patient populations.

*<p>Time to distant metastasis ranged from 9.1–225.7 months, with a median of 48.1 months.</p

<i>SBSN</i> is important in maintaining invasion and metastatic capability in SACC83.

<p>Matrigel invasion assay was performed with scramble control and three types of <i>SBSN</i> shRNA stable clones made from SACC83, as indicated by scramble, shRNA 1, shRNA 2, and shRNA 3. Representative photos of whole transwell membranes are pictured at 4× magnification; the inset pictures were taken at 20× magnification at randomly selected central locations. This metastasis analysis was performed in triplicate.</p

Novel Insight into Mutational Landscape of Head and Neck Squamous Cell Carcinoma

<div><p>Development of head and neck squamous cell carcinoma (HNSCC) is characterized by accumulation of mutations in several oncogenes and tumor suppressor genes. We have formerly described the mutation pattern of HNSCC and described NOTCH signaling pathway alterations. Given the complexity of the HNSCC, here we extend the previous study to understand the overall HNSCC mutation context and to discover additional genetic alterations. We performed high depth targeted exon sequencing of 51 highly actionable cancer-related genes with a high frequency of mutation across many cancer types, including head and neck. DNA from primary tumor tissues and matched normal tissues was analyzed for 37 HNSCC patients. We identified 26 non-synonymous or stop-gained mutations targeting 11 of 51 selected genes. These genes were mutated in 17 out of 37 (46%) studied HNSCC patients. Smokers harbored 3.2-fold more mutations than non-smokers. Importantly, TP53 was mutated in 30%, NOTCH1 in 8% and FGFR3 in 5% of HNSCC. HPV negative patients harbored 4-fold more TP53 mutations than HPV positive patients. These data confirm prior reports of the HNSCC mutational profile. Additionally, we detected mutations in two new genes, CEBPA and FES, which have not been previously reported in HNSCC. These data extend the spectrum of HNSCC mutations and define novel mutation targets in HNSCC carcinogenesis, especially for smokers and HNSCC without HPV infection.</p></div

Clinical characteristics of the cohort.

<p>Clinical characteristics of the cohort.</p

Complete list of point mutations among the 11 cancer genes sequenced in the 37 HNSCC tumor tissues.

<p>Complete list of point mutations among the 11 cancer genes sequenced in the 37 HNSCC tumor tissues.</p

The 51 cancer genes selected for targeted or whole exon sequencing.

<p>The 51 cancer genes selected for targeted or whole exon sequencing.</p

Genetic alterations in 37 HNSCC tumors.

<p>Heat-map representation of individual mutations present in a series of 37 HNSCC tumors, presented in columns. Mutation events are represented by black color. Left, Mutated genes, asterisks indicate genes characterized by whole-exon sequencing. Novel genes mutated in HNSCC are labeled by bold font. Right, mutation rate for each gene. Genes are ranked by mutation rate. Bottom, number of mutations per tumor sample. Note, that two patients have two mutations in the same gene TP53 gene: X16 and X27 (with number 2 on the heat-map). The smoking status of tumor patients is identified as: S – for smokers, NS – for never smoked patients, ND – not determined. The HPV status of tumor patients is identified as: “+” for HPV-positive patients and “-” for HPV-negative patients.</p