Supplementary Figures with legends- Supplementary Figures S1 through S7. (1) Fig.S1 shows the data about transcriptional regulation of INCENP by MYCN in NB cells and the results of Kaplan-Meier survival probability analysis based on the expression of INCENP in NB tumors in the Kocak cohort. (2) Fig.S2 shows the results of rescue experiment of INCENP knockdown by overexpressing a siRNA resistant form of INCENP and the growth inhibition effects in additional NB cell lines (KCNR and Kelly cells) upon INCENP depletion. (3) Fig.S3 shows the results of colony formation assay and soft agar assay performed using dox inducible shCTRL NB cells. (4) Fig.S4 shows the xenograft growth of shCTRL NB tumors and the murine survival of shCTRL tumor-bearing mice upon dox treatment. (5) Fig. S5 shows the polyploidy phenotypes in SY5Y, 293T, ARPE19 and HeLa cells. (6) Fig.S6 shows the induction of DNA damage response in shINCENP NB cells and the induction of apoptosis in both shINCENP NB cells and INCENP-depleted 293T, ARPE19 an HeLa cells. (7) Fig.S7 shows the cellular senescence phenotypes upon Aurora B inhibition or shINCENP mediated INCENP silencing in NB cells.
ARTICLE ABSTRACT
Chromosomal passenger complex (CPC) has been demonstrated to be a potential target of cancer therapy by inhibiting Aurora B or survivin in different types of cancer including neuroblastoma. However, chemical inhibition of either Aurora B or survivin does not target CPC specifically due to off-target effects or CPC-independent activities of these two components. In a previous chromatin-focused siRNA screen, we found that neuroblastoma cells were particularly vulnerable to loss of INCENP, a gene encoding a key scaffolding component of the CPC. In this study, INCENP was highly expressed by neuroblastoma cells, and its expression decreased following retinoic acid–induced neuroblastoma differentiation. Elevated levels of INCENP were significantly associated with poor prognosis in primary tumors of neuroblastoma patients with high-risk disease. Genetic silencing of INCENP reduced the growth of both MYCN–wild-type and MYCN-amplified neuroblastoma cell lines in vitro and decreased the growth of neuroblastoma xenografts in vivo, with significant increases in murine survival. Mechanistically, INCENP depletion suppressed neuroblastoma cell growth by inducing polyploidization, apoptosis, and senescence. In most neuroblastoma cell lines tested in vitro, apoptosis was the primary cell fate after INCENP silencing due to induction of DNA damage response and activation of the p53–p21 axis. These results confirm that CPC is a therapeutic target in neuroblastoma, and targeting INCENP is a novel way to disrupt the activity of CPC and inhibit tumor progression in neuroblastoma.
Dysregulation of INCENP contributes to neuroblastoma tumorigenesis and targeting INCENP presents a novel strategy to disrupt the activity of chromosomal passenger complex and inhibit neuroblastoma progression.
