Sandra Muñoz-Galván

Distinct Roles of Mus81, Yen1, Slx1-Slx4, and Rad1 Nucleases in the Repair of Replication-Born Double-Strand Breaks by Sister Chromatid Exchange

ABSTRACT Most spontaneous DNA double-strand breaks (DSBs) arise during replication and are repaired by homologous recombination (HR) with the sister chromatid. Many proteins participate in HR, but it is often difficult to determine their in vivo functions due to the existence of alternative pathways. Here we take advantage of an in vivo assay to assess repair of a specific replication-born DSB by sister chromatid recombination (SCR). We analyzed the functional relevance of four structure-selective endonucleases (SSEs), Yen1, Mus81-Mms4, Slx1-Slx4, and Rad1, on SCR in Saccharomyces cerevisiae. Physical and genetic analyses showed that ablation of any of these SSEs leads to a specific SCR decrease that is not observed in general HR. Our work suggests that Yen1, Mus81-Mms4, Slx4, and Rad1, but not Slx1, function independently in the cleavage of intercrossed DNA structures to reconstitute broken replication forks via HR with the sister chromatid. These unique effects, which have not been detected in other studies unless double mutant combinations were used, indicate the formation of distinct alternatives for the repair of replication-born DSBs that require specific SSEs.

Histone H3K56 Acetylation, Rad52, and Non-DNA Repair Factors Control Double-Strand Break Repair Choice with the Sister Chromatid

DNA double-strand breaks (DSBs) are harmful lesions that arise mainly during replication. The choice of the sister chromatid as the preferential repair template is critical for genome integrity, but the mechanisms that guarantee this choice are unknown. Here we identify new genes with a specific role in assuring the sister chromatid as the preferred repair template. Physical analyses of sister chromatid recombination (SCR) in 28 selected mutants that increase Rad52 foci and inter-homolog recombination uncovered 8 new genes required for SCR. These include the SUMO/Ub-SUMO protease Wss1, the stress-response proteins Bud27 and Pdr10, the ADA histone acetyl-transferase complex proteins Ahc1 and Ada2, as well as the Hst3 and Hst4 histone deacetylase and the Rtt109 histone acetyl-transferase genes, whose target is histone H3 Lysine 56 (H3K56). Importantly, we use mutations in H3K56 residue to A, R, and Q to reveal that H3K56 acetylation/deacetylation is critical to promote SCR as the major repair mechanism for replication-born DSBs. The same phenotype is observed for a particular class of rad52 alleles, represented by rad52-C180A, with a DSB repair defect but a spontaneous hyper-recombination phenotype. We propose that specific Rad52 residues, as well as the histone H3 acetylation/deacetylation state of chromatin and other specific factors, play an important role in identifying the sister as the choice template for the repair of replication-born DSBs. Our work demonstrates the existence of specific functions to guarantee SCR as the main repair event for replication-born DSBs that can occur by two pathways, one Rad51-dependent and the other Pol32-dependent. A dysfunction can lead to genome instability as manifested by high levels of homolog recombination and DSB accumulation.

The essential role of PIM kinases in sarcoma growth and bone invasion

PIM kinases are a family of serine/threonine kinases composed of three different isoforms (PIM1, PIM 2 and PIM 3) that are highly homologous. Their expression is mediated by the JAK/STAT signalling pathway, providing survival and cell cycle transition signals. PIM kinases are heavily targeted for anticancer drug discovery. However, very little is known about the relative contribution of the different isoforms to the tumourigenesis process in vivo, and how their individual inhibition might affect tumour growth. Taking advantage of genetically modified mice, we explored whether the inhibition of specific isoforms is required to prevent sarcomas induced by 3-methylcholanthrene carcinogenic treatment. We found that absence of Pim2 and Pim3 greatly reduced sarcoma growth to a similar extent to the absence of all three isoforms. This model of sarcoma generally produces bone invasion by the tumour cells. Lack of Pim2 and Pim3 reduced tumour-induced bone invasion by 70%, which is comparable with the reduction of tumour-induced bone invasion in the absence of all three isoforms. Similar results were obtained in mouse embryonic fibroblasts (MEFs) derived from these knockout (KO) mice, where double Pim2/3 KO MEFs already showed reduced proliferation and were resistant to oncogenic transformation by the RAS oncogene. Our data also suggest an important role of Gsk3β phosphorylation in the process of tumourigenesis.

Conditional transgenic expression of PIM1 kinase in prostate induces inflammation-dependent neoplasia.

The Pim proteins are a family of highly homologous protein serine/threonine kinases that have been found to be overexpressed in cancer. Elevated levels of Pim1 kinase were first discovered in human leukemia and lymphomas. However, more recently Pim1 was found to be increased in solid tumors, including pancreatic and prostate cancers, and has been proposed as a prognostic marker. Although the Pim kinases have been identified as oncogenes in transgenic models, they have weak transforming abilities on their own. However, they have been shown to greatly enhance the ability of other genes or chemical carcinogens to induce tumors. To explore the role of Pim1 in prostate cancer, we generated conditional Pim1 transgenic mice, expressed Pim1 in prostate epithelium, and analyzed the contribution of PIM1 to neoplastic initiation and progression. Accordingly, we explored the effect of PIM1 overexpression in 3 different settings: upon hormone treatment, during aging, and in combination with the absence of one Pten allele. We have found that Pim1 overexpression increased the severity of mouse prostate intraepithelial neoplasias (mPIN) moderately in all three settings. Furthermore, Pim1 overexpression, in combination with the hormone treatment, increased inflammation surrounding target tissues leading to pyelonephritis in transgenic animals. Analysis of senescence induced in these prostatic lesions showed that the lesions induced in the presence of inflammation exhibited different behavior than those induced in the absence of inflammation. While high grade prostate preneoplastic lesions, mPIN grades III and IV, in the presence of inflammation did not show any senescence markers and demonstrated high levels of Ki67 staining, untreated animals without inflammation showed senescence markers and had low levels of Ki67 staining in similar high grade lesions. Our data suggest that Pim1 might contribute to progression rather than initiation in prostate neoplasia.

Efficacy of CDK4 inhibition against sarcomas depends on their levels of CDK4 and p16ink4 mRNA

Sarcomas are malignant tumors accounting for a high percentage of cancer morbidity and mortality in children and young adults. Surgery and radiation therapy are the accepted treatments for most sarcomas; however, patients with metastatic disease are treated with systemic chemotherapy. Many tumors display marginal levels of chemoresponsiveness and new treatment approaches are needed. Deregulation of the G1 checkpoint is crucial for various oncogenic transformation processes, suggesting that many cancer cell types depend on CDK4/6 activity. Thus, CDK4/6 activity appears to represent a promising therapeutic target for cancer treatment. In the present work, we explore the efficacy of CDK4 inhibition using palbociclib (PD0332991), a highly selective inhibitor of CDK4/6, in a panel of sarcoma cell lines and sarcoma tumor xenografts (PDXs). Palbociclib induces senescence in these cell lines and the responsiveness of these cell lines correlated with their levels of CDK4 mRNA. Palbociclib is also active in vivo against sarcomas displaying high levels of CDK4 but not against sarcomas displaying low levels of CDK4 and high levels of p16ink4a. The analysis of tumors growing after palbociclib showed a clear decrease in the CDK4 levels, indicating that clonal selection occurred in these treated tumors. In summary, our data support the efficacy of CDK4 inhibitors against sarcomas displaying increased CDK4 levels, particularly fibrosarcomas and MPNST. Our results also suggest that high levels of p16ink4a may indicate poor efficacy of CDK4 inhibitors.

Oxidation of anticancer Pt(II) complexes with monodentate phosphane ligands: towards stable but active Pt(IV) prodrugs

The synthesis, characterization and cytotoxicity studies of two novel platinum(IV) complexes, trans-PtCl4(dma)(PPh3), 1, and trans-PtCl4(ipa)(PPh3), 2, where dma is dimethylamine and ipa is isopropylamine, have been carried out. Both complexes contain aliphatic amines trans to phosphane ligands as a good alternative to take advantage of the phosphane group lipophilicity and the stability of platinum(IV) to obtain more effective drugs. Moreover, the complexes are stable in solution and such stability allowed their antitumoral action and DNA interaction to be checked and proved.

Competing roles of DNA end resection and non-homologous end joining functions in the repair of replication-born double-strand breaks by sister-chromatid recombination

While regulating the choice between homologous recombination and non-homologous end joining (NHEJ) as mechanisms of double-strand break (DSB) repair is exerted at several steps, the key step is DNA end resection, which in Saccharomyces cerevisiae is controlled by the MRX complex and the Sgs1 DNA helicase or the Sae2 and Exo1 nucleases. To assay the role of DNA resection in sister-chromatid recombination (SCR) as the major repair mechanism of spontaneous DSBs, we used a circular minichromosome system for the repair of replication-born DSBs by SCR in yeast. We provide evidence that MRX, particularly its Mre11 nuclease activity, and Sae2 are required for SCR-mediated repair of DSBs. The phenotype of nuclease-deficient MRX mutants is suppressed by ablation of Yku70 or overexpression of Exo1, suggesting a competition between NHEJ and resection factors for DNA ends arising during replication. In addition, we observe partially redundant roles for Sgs1 and Exo1 in SCR, with a more prominent role for Sgs1. Using human U2OS cells, we also show that the competitive nature of these reactions is likely evolutionarily conserved. These results further our understanding of the role of DNA resection in repair of replication-born DSBs revealing unanticipated differences between these events and repair of enzymatically induced DSBs.

MAP17 (PDZKIP1) Expression Determines Sensitivity to the Proteasomal Inhibitor Bortezomib by Preventing Cytoprotective Autophagy and NFκB Activation in Breast Cancer

MAP17 is a small nonglycosylated membrane protein that is overexpressed in a high percentage of carcinomas. High levels of MAP17 enhance the tumorigenic properties of tumor cells by increasing oxidative stress, which is dependent on Na+-coupled cotransport. Here, we show that MAP17 is associated with proteins involved in protein degradation and that proteasome inhibition induces autophagy. To analyze whether MAP17 could also alter this process, we used the proteasome inhibitor bortezomib (Velcade, PS-341), which is approved for the treatment of multiple myeloma and mantle cell lymphoma, although it has a high rate of resistance emergence and poor efficacy in solid tumors. We provide evidence that bortezomib induces a cytoprotective effect by activating autophagy and NFκB nuclear translocation, responses that are repressed in the presence of high levels of MAP17 both in vitro and in vivo. Indeed, patients with multiple myeloma treated with bortezomib showed higher response rates and a longer time to progression associated with increased levels of MAP17 expression. The MAP17-induced sensitivity to bortezomib is dependent on the oxidative status of the cells and the activity of Na+-coupled transporters because treatment with antioxidants or the inhibitor furosemide restores the cytoprotective activity induced by bortezomib. Therefore, bortezomib induces a prosurvival response through cytoprotective autophagy and NFκB nuclear translocation, which is repressed by high levels of MAP17. We propose that the levels of MAP17 could be used as a prognostic marker to predict the response to bortezomib in hematologic malignancies and in other tissues that are not commonly responsive to the drug. Mol Cancer Ther; 14(6); 1454–65. ©2015 AACR.

Spinophilin Loss Correlates with Poor Patient Prognosis in Advanced Stages of Colon Carcinoma

Purpose: The genomic region 17q21 is frequently associated with microsatellite instability and LOH in cancer, including gastric and colorectal carcinomas. This region contains several putative tumor suppressor genes, including Brca1, NM23, prohibitin, and spinophilin (Spn, PPP1R9B, neurabin II). The scaffold protein Spn is one of the regulatory subunits of phosphatase-1 (PP1) that targets PP1 to distinct subcellular locations and couples PP1 to its target. Thus, Spn may alter cell-cycle progression via the regulation of the phosphorylation status of the retinoblastoma protein, a direct target of PP1. Therefore, we analyzed whether Spn levels were reduced in colorectal carcinomas and whether Spn levels correlated with prognosis or response to therapy. Experimental Design: By means of immunohistochemistry or quantitative PCR, we studied the levels of Spn in stages II, III, and IV colorectal carcinoma tumors and correlated to other clinicopathologic features as well as prognosis or response to therapy. Results: Spn was lost in a percentage of human gastric, small intestine, and colorectal carcinomas. In patients with colorectal carcinoma, tumoral Spn downregulation correlated with a more aggressive histologic phenotype (poorer tumor differentiation and higher proliferative Ki67 index). Consistent with this observation, lower Spn protein expression levels were associated with faster relapse and poorer survival in patients with stage III colorectal carcinoma, particularly among those receiving adjuvant fluoropyrimidine therapy. We validated this result in an independent cohort of patients with metastatic colorectal carcinoma treated with standard chemotherapy. Although patients that achieved an objective tumor response exhibited Spn levels similar to nontumoral tissue, nonresponding patients showed a significant reduction in Spn mRNA levels. Conclusions: Our data suggest that Spn downregulation contributes to a more aggressive biologic behavior, induces chemoresistance, and is associated with a poorer survival in patients with advanced stages of colorectal carcinoma. Clin Cancer Res; 19(14); 3925–35. ©2013 AACR.

Loss of the tumor suppressor spinophilin (PPP1R9B) increases the cancer stem cell population in breast tumors

The spinophilin (Spn, PPP1R9B) gene is located at 17q21.33, a region frequently associated with microsatellite instability and loss of heterozygosity, especially in breast tumors. Spn is a regulatory subunit of phosphatase1a (PP1), which targets the catalytic subunit to distinct subcellular locations. Spn downregulation reduces PPP1CA activity against the retinoblastoma protein, pRb, thereby maintaining higher levels of phosphorylated pRb. This effect contributes to an increase in the tumorigenic properties of cells in certain contexts. Here, we explored the mechanism of how Spn downregulation contributes to the malignant phenotype and poor prognosis in breast tumors and found an increase in the stemness phenotype. Analysis of human breast tumors showed that Spn mRNA and protein are reduced or lost in 15% of carcinomas, correlating with a worse prognosis, a more aggressive tumor phenotype and triple-negative tumors, whereas luminal tumors showed high Spn levels. Downregulation of Spn by shRNA increased the stemness properties along with the expression of stem-related genes (Sox2, KLF4, Nanog and OCT4), whereas ectopic overexpression of Spn cDNA reduced these properties. Breast tumor stem cells appeared to have low levels of Spn mRNA, and Spn loss correlated with increased stem-like cell appearance in breast tumors as indicated by an increase in CD44+/CD24- cells. A reduction of the levels of PPP1CA mimicked the cancer stem-like cell phenotype of Spn downregulation, suggesting that the mechanism of Spn involves PP1a. These increased cancer stem cell-like properties with reduced Spn might account for the malignant phenotype observed in Spn-loss tumors and may contribute to a worse patient prognosis.