April 21, 2024

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How do genetic mutations cause tissue-specific cancers?

How do genetic mutations cause tissue-specific cancers?


How do genetic mutations cause tissue-specific cancers? Renal lineage factor PAX8 controls oncogenic signaling in kidney cancer.

How genetic mutations contribute to tissue-specific cancer phenotypes remains a fundamental open question in cancer biology.

Somatic mutations in most cancer driver genes are detected in only a few tumor types, and inherited cancer susceptibility alleles are often associated with cancer risk in a tissue-specific manner.

The tissue origin of cancer suggests that transcriptional networks that define cellular states may be critical for oncogenic processes. Lineage transcription factors (TFs) , such as SOX10 in melanoma, are often required for cancer cell survival and proliferation.

In addition, the most common VHL loss-of-function mutation in human clear cell renal cell carcinoma ( ccRCC ) is extremely rare in other types of cancer, and loss-of-function VHL can lead to the stabilization of hypoxia-inducible factors HIF1A and HIF2A, which in turn activate their downstream transcription program.

However, it remains unclear whether specific interactions between lineage factors and genetic alterations are required to establish cancer-type-specific oncogenic programs.


On June 8, 2022, the team of Sakari Vanharanta from the University of Cambridge, UK, published an article in the journal Nature entitled The renal lineage factor PAX8 controls oncogenic signalling in kidney cancer.

Analysis of the samples, demonstrating that the kidney lineage transcription factor paired box 8 ( PAX8 ) is the oncogenic signal of two genetic alterations in ccRCC (germline variant rs7948643 at 11q13.3 and somatic inactivation of VHL) Necessary for transduction, emphasizing that transcriptional lineage factors mediate tissue-specific cancer risk associated with somatic and inherited genetic alterations.


How do genetic mutations cause tissue-specific cancers?


We first screened TFs that supported the proliferation of two metastatic ccRCC cell lines (OS-LM1 and 786-M1A) , two factors PAX8 and HNF1B showed strong specificity for ccRCC cell lines.

At the same time, they characterized the nuclear complex occupied by HIF2A by rapid immunoprecipitation mass spectrometry analysis of endogenous proteins (RIME) [1] , in which PAX8 is a member of the HIF2A nuclear interactome.

The 89 proteins in complexes with HIF2A and PAX8 at the same time are nuclear proteins with functions in chromatin remodeling (SWI/SNF complex) or mRNA processing.

ChIP-seq analysis of xenografted ccRCC tumors also revealed a high frequency of co-localization of HIF2A and PAX8 on chromatin, in brief, 43% and 65% of HIF2A bound in 786-M1A and OS-LM1 tumors, respectively Sites showed significant PAX8 binding.



Next, we developed a tumor model that reintroduced HIF2A via a doxycycline-dependent transgene in 786-M1A HIF2A-/- cells, thereby confirming the transcriptomic effect of HIF2A inhibition at different time points by RNA-seq , and identified 175 HIF2A ChIP-seq peaks within the 500-kb region flanking the transcription start site of the 205 strongly HIF2A-dependent transcripts detected. Using a tandem design that effectively inhibits enhancer function, they generated a library of 706 sgRNA pairs targeting these peaks and controls, co-transfected with dCas9-KRAB into 786-M1A cells [2] , and transplanted into 15 NSGs. in mice. Twenty-one constructs targeting the HIF2A-binding enhancer were depleted in tumors, 16 of which showed HIF2A and PAX8 binding.

Among them, enhancer E11:69419 overlapped with one of the strongest ccRCC-specific open chromatin regions in the large clinical ATAC-seq cancer dataset [3] and was significantly activated in ccRCC but not papillary RCC.

E11:69419 is flanked by the genes MYEOV and CCND1 , which encodes cyclin D1, a positive cell cycle regulator that is activated in several cancer types and whose expression is controlled by the VHL-HIF2A pathway.

CRISPRi-mediated suppression of E11:69419 resulted in downregulation of these two genes, and suppression of PAX8 and HIF2A also reduced CCND1 expression.


The most important of the common genetic variants identified by GWAS to be associated with human RCC risk is rs7105934 on chromosome 11q13.3 [4] , this risk haplotype contains E11:69419 and covers the linked SNPs rs7948643 and rs7939721.

Among them, rs7948643 is located at the PAX8 binding site in E11:69419, where the allele T shows a higher affinity than C, which means that the protective allele C can inhibit the binding of PAX8, thereby reducing the oncogenic driver.

E11:69419 activity upstream of CCND1. In addition, depletion of PAX8 also reduced HIF2A binding at E11:69419, whereas depletion of HIF2A did not affect PAX8 binding.


In addition, we found that PAX8 inhibition impairs ccRCC cell proliferation in vitro, suggesting the existence of HIF2A-independent oncogenic PAX8 function .

PAX8 positively regulates HNF1B expression, and reintroduction of exogenous PAX8 or HNF1B rescues the in vitro proliferation defect caused by PAX8 depletion.

Genes down-regulated in PAX8-depleted and HNF1B-depleted cells were identified based on a genome-wide CRISPR–Cas9 screen [5] , only HNF1B and MYC met the criteria, while genes up-regulated after depletion did not show any inhibition of ccRCC proliferation gene.

Increased copy number of MYC and its regulatory regions was associated with ccRCC metastasis, and FISH analysis revealed that metastatic 786-M1A cells carried 6 copies of MYC.

Using a functional CRISPRi screen, we identified eight distal MYC enhancers important for ccRCC proliferation, two of which (E8:128132 and E8:128526) were shown to bind HNF1B and contain the HNF1B motif, in HNF1B and PAX8 Decreased chromatin accessibility was also shown after knockdown.

Thus, cancer-specific 8q21.3-q24.3 expansion in ccRCC cells selects a lineage factor-dependent physiological program supporting MYC expression and proliferation already present in normal renal epithelial cells.


Collectively, this work identified rs7948643 as a common genetic variant associated with the most important renal cancer risk locus on chromosome 11q13.3, rs7105934, belonging to the PAX8 binding site within E11:69419, a pair of PAX8 and HIF2A The requirement for E11:69419 activity and the strong association of rs7948643 with ccRCC but not papillary RCC support a model that differences in PAX8 binding at rs7948643 are responsible for the increased risk of ccRCC associated with this locus.

In conclusion, this work provides functional insights into the mechanisms governing the interplay between genetic and somatic genetic alterations and developmental lineage factors to determine the risk of cancer, especially ccRCC. PAX8 supports the expression of two canonical oncogenes , CCND1 and MYC , and mouse kidneys can tolerate genetic inactivation of Pax8 , suggesting that PAX8 may be a potentially viable therapeutic target in ccRCC.












Original link: https://doi.org/10.1038/s41586-022-04809-8 Publisher: Eleven

1. Papachristou, E. K. et al. A quantitative mass spectrometry-based approach to monitor the dynamics of endogenous chromatin-associated protein complexes. Nat. Commun. 9, 2311 (2018).

2. Larson, M. H. et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat. Protoc. 8, 2180–2196 (2013).

3. Corces, M. R. et al. The chromatin accessibility landscape of primary human cancers. Science 362, eaav1898 (2018).

4. Purdue, M. P. et al. Genome-wide association study of renal cell carcinoma identifies two susceptibility loci on 2p21 and 11q13.3. Nat. Genet. 43, 60–65 (2011).

5. Dempster, J. M. et al. Extracting biological insights from the Project Achilles Genome-Scale CRISPR screens in cancer cell lines. Preprint at bioRxiv https://doi. org/10.1101/720243 (2019).

How do genetic mutations cause tissue-specific cancers?

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