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Wilms Tumor: insights into the role of integrins driving cancer stem cells self-renewal vs. differentiation
Astgik Petrosyan, PhD1, Valentina Villani, PhD1, Paola Aguiari, PhD1, Min Y. Madhi, MS1, Matthew E. Thornton, MS2, Brendan H. Grubbs, MD2, Kevin Lemley, MD, PhD1, Roger De Filippo, MD1, Sargis Sedrakyan, PhD1, Gregory Shackleford, PhD1, Anat Erdreich-Epstein, MD1, Stefano Da Sacco, PhD1, Laura Perin, PhD3.
1Children's Hospital Los Angeles, Los Angeles, CA, USA, 2University of Southern California, Los Angeles, CA, USA, 3Children's Hospital Los Angeles, los angeles, CA, USA.

BACKGROUND: Wilms Tumor (WT) is the most common pediatric renal cancer. Growing evidence links WT to aberrant nephrogenesis. While studies highlighted the genetic complexity of WT, little is known about the molecular mechanisms that regulate WT development. Here we report that uncommitted nephrogenic progenitor (NP) expressing SIX2 and CITED1 (the master transcription factors regulating renal development) present characteristics of cancer stem cells (CSCs) and are the ones driving WT. We have also studied the role of integrins in these NP in regulating WT development.
METHODS: WT and human fetal kidney (hFK) samples were histologically analyzed, digested to single-cell suspension, incubated with Smartflare-probe to isolate SIX2+CITED1+ cells, and processed for RNA-seq, single-cell RNA-seq and spatial transcriptomics. Xenografts of WT-NP and hFK-NP were generated and tumor formation was assessed. Analyses of mechanisms that regulate self-renewal vs. differentiation were performed in vitro and in vivo. Knockdown with miREs against SIX2 and CITED1 was performed on WT-NP and processed for RNA-seq.
RESULTS: By comparing NP from different WT subtypes and NP from hFK we identified that cells expressing SIX2 and CITED1 fulfill CSC criteria, reliably recapitulating WT in transplantation studies. We showed that self-renewal vs. differentiation of SIX2+CITED1+ WT CSC is regulated by the interplay between integrins ITGB1 and ITGB4. WT transplantation studies show that blocking ITGB1 or ITGB4 leads to higher number of SIX2+CITED1+ cells in the xenografts. Knockdown of SIX2 and CITED1 increased expression of kidney differentiation markers LHX1, WNT7B, PODXL, MECOM, reduced expression of nephrogenic markers MEOX1, TMEM100, EYA1, MAYFB, and increased expression of ITGB1, ITGB4, and LAMA5.
CONCLUSIONS: These studies define SIX2+CITED1+ cells as the nephrogenic CSC of WT, where ITGB1 and ITGB4 interplay may play a role in self-renewal vs. differentiation and serve as a potential target for new strategies to treat WT.


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