RESUMEN
Spalt-like proteins are Zinc finger transcription factors from Caenorhabditis elegans to vertebrates, with critical roles in development. In vertebrates, four paralogues have been identified (SALL1-4), and SALL2 is the family's most dissimilar member. SALL2 is required during brain and eye development. It is downregulated in cancer and acts as a tumor suppressor, promoting cell cycle arrest and cell death. Despite its critical functions, information about SALL2 regulation is scarce. Public data indicate that SALL2 is ubiquitinated and phosphorylated in several residues along the protein, but the mechanisms, biological consequences, and enzymes responsible for these modifications remain unknown. Bioinformatic analyses identified several putative phosphorylation sites for Casein Kinase II (CK2) located within a highly conserved C-terminal PEST degradation motif of SALL2. CK2 is a serine/threonine kinase that promotes cell proliferation and survival and is often hyperactivated in cancer. We demonstrated that CK2 phosphorylates SALL2 residues S763, T778, S802, and S806 and promotes SALL2 degradation by the proteasome. Accordingly, pharmacological inhibition of CK2 with Silmitasertib (CX-4945) restored endogenous SALL2 protein levels in SALL2-deficient breast MDA-MB-231, lung H1299, and colon SW480 cancer cells. Silmitasertib induced a methuosis-like phenotype and cell death in SW480 cells. However, the phenotype was significantly attenuated in CRISPr/Cas9-mediated SALL2 knockout SW480 cells. Similarly, Sall2-deficient tumor organoids were more resistant to Silmitasertib-induced cell death, confirming that SALL2 sensitizes cancer cells to CK2 inhibition. We identified a novel CK2-dependent mechanism for SALL2 regulation and provided new insights into the interplay between these two proteins and their role in cell survival and proliferation.
Asunto(s)
Quinasa de la Caseína II , Neoplasias del Colon , Animales , Humanos , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Neoplasias del Colon/genética , Línea Celular TumoralRESUMEN
Colorectal cancer (CRC) is one of the most common malignant neoplasms and the second leading cause of death from tumors worldwide. Therefore, there is a great need to study new therapeutical strategies, such as effective immunotherapies against these malignancies. Unfortunately, many CRC patients do not respond to current standard immunotherapies, making it necessary to search for adjuvant treatments. Histone deacetylase 6 (HDAC6) is involved in several processes, including immune response and tumor progression. Specifically, it has been observed that HDAC6 is required to activate the Signal Transducer and Activator of Transcription 3 (STAT3), a transcription factor involved in immunogenicity, by activating different genes in these pathways, such as PD-L1. Over-expression of immunosuppressive pathways in cancer cells deregulates T-cell activation. Therefore, we focused on the pharmacological inhibition of HDAC6 in CRC cells because of its potential as an adjuvant to avoid immunotolerance in immunotherapy. We investigated whether HDAC6 inhibitors (HDAC6is), such as Nexturastat A (NextA), affected STAT3 activation in CRC cells. First, we found that NextA is less cytotoxic than the non-selective HDACis panobinostat. Then, NextA modified STAT3 and decreased the mRNA and protein expression levels of PD-L1. Importantly, transcriptomic analysis showed that NextA treatment affected the expression of critical genes involved in immunomodulatory pathways in CRC malignancies. These results suggest that treatments with NextA reduce the functionality of STAT3 in CRC cells, impacting the expression of immunomodulatory genes involved in the inflammatory and immune responses. Therefore, targeting HDAC6 may represent an interesting adjuvant strategy in combination with immunotherapy.
Asunto(s)
Antígeno B7-H1 , Neoplasias Colorrectales , Humanos , Histona Desacetilasa 6/metabolismo , Antígeno B7-H1/metabolismo , Factor de Transcripción STAT3/metabolismo , Inmunidad , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patologíaRESUMEN
The Sall2 transcription factor is deregulated in several cancers; however, little is known about its cellular functions, including its target genes. Recently, we demonstrated that p53 directly regulates Sall2 expression under genotoxic stress. Here, we investigated the role of Sall2 in the context of cellular response to genotoxic stress. In addition, we further examined the Sall2-p53 relationship during genotoxic stress in primary mouse embryo fibroblasts (MEFs), which are derived from Sall2 knockout mice separately, or in combination with the p53ERTAM knock-in mice. We found that the levels of Sall2 mRNA and protein are dynamically modulated in response to doxorubicin. At early times of stress, Sall2 is downregulated, but increases under extension of the stress in a p53-independent manner. Based on caspase-3/7 activities, expression of cleaved poly (ADP-ribose) polymerase, expression of cleaved caspase-3 and induction of proapoptotic proteins, Sall2 expression was correlated with cellular apoptosis. Consequently, Sall2-/- MEFs have decreased apoptosis, which relates with increased cell viability in response to doxorubicin. Importantly, Sall2 was required for apoptosis even in the presence of fully activated p53. Searching for putative Sall2 targets that could mediate its role in apoptosis, we identified proapoptotic NOXA/PMAIP1 (phorbol-12-myristate-13-acetate-induced protein 1). We demonstrated that Sall2 positively regulates Noxa promoter activity. Conserved putative Sall2-binding sites at the NOXA promoter were validated in vitro by electrophoretic mobility shift assay and in vivo by ChIP experiments, identifying NOXA as a novel Sall2 target. In agreement, induction of Noxa protein and mRNA in response to doxorubicin was significantly decreased in Sall2-/- MEFs. In addition, studies in leukemia Jurkat T cells support the existence of the Sall2/Noxa axis, and the significance of this axis on the apoptotic response to doxorubicin in cancer cells. Our study highlights the relevance of Sall2 in the apoptotic response to extended genotoxic stress, which is important for understanding its role in normal physiology and disease.