Oncogenic Ect2 signaling regulates rRNA synthesis in NSCLC

Abstract

The Rho GTPase family members Rac1, Cdc42 and RhoA play key contributory roles in the transformed phenotype of human cancers. Epithelial Cell Transforming Sequence 2 (Ect2), a guanine nucleotide exchange factor (GEF) for these Rho GTPases, has also been implicated in a variety of human cancers. We have shown that Ect2 is frequently overexpressed in both major forms of non-small cell lung cancer (NSCLC), lung adenocarcinoma (LADC) and lung squamous cell carcinoma (LSCC), which together make up approximately 70% of all lung cancer diagnoses. Furthermore, we have found that Ect2 is required for multiple aspects of the transformed phenotype of NSCLC cells including transformed growth and invasion in vitro and tumorigenesis in vivo. More recently, we showed that a major mechanism by which Ect2 drives KRAS-mediated LADC transformation is by regulating rRNA (rRNA) synthesis. However, it remains unclear whether Ect2 plays a similar role in ribosome biogenesis in LSCC. Here we demonstrate that Ect2 expression correlates positively with expression of ribosome biogenesis genes and with pre-ribosomal 45S RNA abundance in primary LSCC tumors. Furthermore, we demonstrate that Ect2 functionally regulates rRNA synthesis in LSCC cells. Based on these data, we propose that inhibition of Ect2-mediated nucleolar signaling holds promise as a potential therapeutic strategy for improved treatment of both LADC and LSCC.

Keywords: Ect2; non-small cell lung cancer; rRNA synthesis; ribosome biogenesis.

Figure 1.

Cytoplasmic and nuclear Ect2 signaling in NSCLC. Cytoplasmic Ect2 binds to the PKCι-Par6α complex and activates a Rac1/Pak/Mek/Erk signaling axis that drives transformation in NSCLC cells.^14,25 Nuclear Ect2 activates rRNA synthesis by binding the nucleolar transcription factor upstream binding factor 1 (UBF1) on rDNA promoters and recruiting Rac1 and its downstream effector nucleophosmin (NPM) to rDNA.¹⁰

Figure 2.

Ect2 regulates 45S rRNA synthesis in LSCC. (A) Co-occurrence analysis of the TCGA LSCC data set (501 cases) for expression of ribosomal processing genes and Rho family GEFs. Significant co-occurrences are shown in red. (See key for p values associated with shade of red). (B) qPCR analysis of Ect2 mRNA and 45S rRNA abundance in human primary lung squamous cell carcinoma (LSCC) cases and normal control lung tissues. The boxes encompass the 25th- 75th percentiles; lines within the boxes indicate the median; lines above and below the boxes indicate the 95th and 5th percentiles, respectively. n = 42; *p < 0.001 and *p < 0.002 for Ect2 and 45S respectively. (C) Scatter plot to assess the correlation between Ect2 mRNA and 45S rRNA in primary LSCC tumors (n = 42; R² = 0.77). qPCR of NT and Ect2 KD H1703, ChagoK-1, H520, LUDLU-1, Oka-C-1 and, HARA cells for (D) Ect2 mRNA and (E) 45S rRNA. Results represent the mean ± SEM; n = 3; *p < 0.0008 compared with NT. (F) Soft agar colony formation to assess transformed growth of LSCC Ect2 KD cells. Results represent the mean +/− SEM; n = 4. * p < 1 × 10⁻⁵ compared with NT cells.

Figure 3.

ECT2 is frequently amplified and overexpressed in multiple human tumor types. (A) Frequency of ECT2 genetic alterations in 29 human tumor types. ECT2 is frequently amplified in many human tumor types. (B) Ect2 is overexpressed in multiple human tumor types. Ect2 mRNA abundance correlates positively with ECT2 amplification in some tumor types, however some tumors that do not exhibit frequent ECT2 amplification express elevated Ect2 mRNA indicating that multiple mechanisms contribute to tumor-specific overexpression of Ect2.