SALL1 functions as a tumor suppressor in breast cancer by regulating cancer cell senescence and metastasis through the NuRD complex.

BACKGROUND: SALL1 is a multi-zinc finger transcription factor that regulates organogenesis and stem cell development, but the role of SALL1 in tumor biology and tumorigenesis remains largely unknown. METHODS: We analyzed SALL1 expression levels in human and murine breast cancer cells as well as cancer tissues from different types of breast cancer patients. Using both in vitro co-culture system and in vivo breast tumor models, we investigated how SALL1 expression in breast cancer cells affects tumor cell growth and proliferation, metastasis, and cell fate. Using the gain-of function and loss-of-function strategies, we dissected the molecular mechanism responsible for SALL1 tumor suppressor functions. RESULTS: We demonstrated that SALL1 functions as a tumor suppressor in breast cancer, which is significantly down-regulated in the basal like breast cancer and in estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2) triple negative breast cancer patients. SALL1 expression in human and murine breast cancer cells inhibited cancer cell growth and proliferation, metastasis, and promoted cell cycle arrest. Knockdown of SALL1 in breast cancer cells promoted cancer cell growth, proliferation, and colony formation. Our studies revealed that tumor suppression was mediated by recruitment of the Nucleosome Remodeling and Deacetylase (NuRD) complex by SALL1, which promoted cancer cell senescence. We further demonstrated that the mechanism of inhibition of breast cancer cell growth and invasion by SALL1-NuRD depends on the p38 MAPK, ERK1/2, and mTOR signaling pathways. CONCLUSION: Our studies indicate that the developmental control gene SALL1 plays a critical role in tumor suppression by recruiting the NuRD complex and thereby inducing cell senescence in breast cancer cells.

Sall1 balances self-renewal and differentiation of renal progenitor cells.

The formation of the proper number of functional nephrons requires a delicate balance between renal progenitor cell self-renewal and differentiation. The molecular factors that regulate the dramatic expansion of the progenitor cell pool and differentiation of these cells into nephron precursor structures (renal vesicles) are not well understood. Here we show that Sall1, a nuclear transcription factor, is required to maintain the stemness of nephron progenitor cells. Transcriptional profiling of Sall1 mutant cells revealed a striking pattern, marked by the reduction of progenitor genes and amplified expression of renal vesicle differentiation genes. These global changes in gene expression were accompanied by ectopic differentiation at E12.5 and depletion of Six2+Cited1+ cap mesenchyme progenitor cells. These findings highlight a novel role for Sall1 in maintaining the stemness of the progenitor cell pool by restraining their differentiation into renal vesicles.

Conditional expression of Wnt9b in Six2-positive cells disrupts stomach and kidney function.

During kidney development, canonical Wnt signaling activates differentiation, while the transcription factor Six2 maintains the progenitor pool. These opposing signals help to regulate nephron formation and ensure the full complement of nephrons are formed. Since these two factors control differing fates in kidney mesenchyme, we hypothesized that overexpression of Wnt9b in Six2-expressing cells would disrupt kidney formation and may alter cell differentiation decisions in other tissues. We created a transgenic mouse that conditionally expressed the canonical Wnt ligand in the developing kidney, Wnt9b. The transgene is activated by cre recombinase and expresses GFP. We first tested its biological activity using Hoxb7-cre and found that transgenic Wnt9b was capable of inducing differentiation genes and of rescuing kidney development in Wnt9b(-/-) homozygous deficient mice. In contrast, expression of Wnt9b in cells using Six2-cre caused gastrointestinal distress and severe renal failure in adult mice. Transgenic kidneys had numerous cystic tubules and elevated creatinine values (0.652 ± 0.044) compared to wild-type mice (0.119 ± 0.002). These animals also exhibited a malformed pyloric sphincter, duodenogastric reflux, and a transformation of the distal stomach into proximal fate. The gene expression changes observed for the Wnt9b:EGFP transgene were compared to a stabilized ß-catenin allele to determine that Wnt9b is activating the canonical Wnt pathway in the tissues analyzed. These results demonstrate that expression of Wnt9b in Six2-positive cells disrupts cell fate decisions in the kidney and the gastrointestinal tract.

Sall1-dependent signals affect Wnt signaling and ureter tip fate to initiate kidney development.

Development of the metanephric kidney depends on precise control of branching of the ureteric bud. Branching events represent terminal bifurcations that are thought to depend on unique patterns of gene expression in the tip compared with the stalk and are influenced by mesenchymal signals. The metanephric mesenchyme-derived signals that control gene expression at the ureteric bud tip are not well understood. In mouse Sall1 mutants, the ureteric bud grows out and invades the metanephric mesenchyme, but it fails to initiate branching despite tip-specific expression of Ret and Wnt11. The stalk-specific marker Wnt9b and the beta-catenin downstream target Axin2 are ectopically expressed in the mutant ureteric bud tips, suggesting that upregulated canonical Wnt signaling disrupts ureter branching in this mutant. In support of this hypothesis, ureter arrest is rescued by lowering beta-catenin levels in the Sall1 mutant and is phenocopied by ectopic expression of a stabilized beta-catenin in the ureteric bud. Furthermore, transgenic overexpression of Wnt9b in the ureteric bud causes reduced branching in multiple founder lines. These studies indicate that Sall1-dependent signals from the metanephric mesenchyme are required to modulate ureteric bud tip Wnt patterning in order to initiate branching.

SALL1 truncated protein expression in Townes-Brocks syndrome leads to ectopic expression of downstream genes.

Mutations in SALL1 lead to the dominant multiorgan congenital anomalies that define Townes-Brocks syndrome (TBS). The majority of these mutations result in premature termination codons that would be predicted to trigger nonsense-mediated decay (NMD) of mutant mRNA and cause haploinsufficiency. Our previous studies using a gene targeted mouse model (Sall1-DeltaZn) suggested that TBS phenotypes are due to expression of a truncated mutant protein, not haploinsufficiency. In this report, we strengthen this hypothesis by showing that expression of the mutant protein alone in transgenic mice is sufficient to cause limb phenotypes that are characteristic of TBS patients. We prove that the same pathogenetic mechanism elucidated in mice is occurring in humans by demonstrating that truncated SALL1 protein is expressed in cells derived from a TBS patient. TBS mutant protein is capable of dominant negative activity that results in ectopic activation of two downstream genes, Nppa and Shox2, in the developing heart and limb. We propose a model for the pathogenesis of TBS in which truncated Sall1 protein causes derepression of Sall-responsive target genes.