Serine hydroxymethyltransferase 2 expression promotes tumorigenesis in rhabdomyosarcoma with 12q13-q14 amplification
Thanh H. Nguyen, … , Wenyue Sun, Frederic G. Barr
Thanh H. Nguyen, … , Wenyue Sun, Frederic G. Barr
Published June 24, 2021
Citation Information: J Clin Invest. 2021;131(15):e138022. https://doi.org/10.1172/JCI138022.
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Research Article Cell biology Oncology

Serine hydroxymethyltransferase 2 expression promotes tumorigenesis in rhabdomyosarcoma with 12q13-q14 amplification

Abstract

The 12q13-q14 chromosomal region is recurrently amplified in 25% of fusion-positive (FP) rhabdomyosarcoma (RMS) cases and is associated with a poor prognosis. To identify amplified oncogenes in FP RMS, we compared the size, gene composition, and expression of 12q13-q14 amplicons in FP RMS with those of other cancer categories (glioblastoma multiforme, lung adenocarcinoma, and liposarcoma) in which 12q13-q14 amplification frequently occurs. We uncovered a 0.2 Mb region that is commonly amplified across these cancers and includes CDK4 and 6 other genes that are overexpressed in amplicon-positive samples. Additionally, we identified a 0.5 Mb segment that is only recurrently amplified in FP RMS and includes 4 genes that are overexpressed in amplicon-positive RMS. Among these genes, only serine hydroxymethyltransferase 2 (SHMT2) was overexpressed at the protein level in an amplicon-positive RMS cell line. SHMT2 knockdown in amplicon-positive RMS cells suppressed growth, transformation, and tumorigenesis, whereas overexpression in amplicon-negative RMS cells promoted these phenotypes. High SHMT2 expression reduced sensitivity of FP RMS cells to SHIN1, a direct SHMT2 inhibitor, but sensitized cells to pemetrexed, an inhibitor of the folate cycle. In conclusion, our study demonstrates that SHMT2 contributes to tumorigenesis in FP RMS and that SHMT2 amplification predicts differential response to drugs targeting this metabolic pathway.

Authors

Thanh H. Nguyen, Prasantha L. Vemu, Gregory E. Hoy, Salah Boudjadi, Bishwanath Chatterjee, Jack F. Shern, Javed Khan, Wenyue Sun, Frederic G. Barr

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Figure 7

Effect of SHMT2 overexpression on Rh41 cell growth and transformation.

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Effect of SHMT2 overexpression on Rh41 cell growth and transformation. (...
(A) SHMT2 protein expression in control or SHMT2-expressing Rh41 cells. Rh30 cells were used for comparison, and GAPDH was used as a loading control. Expression was quantified as described in Figure 5. (B) Measurement of NADPH levels in control or SHMT2-expressing Rh41 cells. Total unfiltered protein concentrations were used for normalization. Data represent mean ± SD of 3 replicates. Student’s t test was used for statistical analysis. (C) IncuCyte growth assay. Data are represented as mean ± SEM of 4 different wells. Multiple t tests (corrected for multiple comparisons using the Šidák-Bonferroni method) were performed for the last 6 time points to assess the significance in growth differences. *P < 0.05, adjusted. (D) Clonogenic assay. 360 cells were seeded in 6 cm dishes, cultured for 3 weeks, then fixed and stained with Giemsa. (E) Focus formation assay: 500 cells were cocultured with 2 × 105 NIH 3T3 fibroblasts for 4 weeks, then fixed and stained with Giemsa. (F) Intramuscular xenograft tumor formation of control or SHMT2-expressing Rh41 cells. All tumors (4 mice per group) were excised when the largest tumor reached the maximum size permitted. (G) Xenograft tumor growth in mice. Tumors (7 mice per group) were measured twice weekly, and data are expressed as mean volume ± SD. Student’s t test was performed to determine the significance of growth differences between the groups. *P < 0.05. Experiments shown in A, C, D, and E were repeated at least 3 times, and representative data are shown. Results shown in F and G were derived from separate independent experiments.