Anna Sablina

RNF4 is required for DNA double-strand break repair in vivo

Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signaling and repair proteins to the sites of DNA lesions. Coordinated protein SUMOylation and ubiquitylation have crucial roles in regulating the dynamic assembly of protein complexes at these sites. However, how SUMOylation influences protein ubiquitylation at DSBs is poorly understood. We show herein that Rnf4, an E3 ubiquitin ligase that targets SUMO-modified proteins, accumulates in DSB repair foci and is required for both homologous recombination (HR) and non-homologous end joining repair. To establish a link between Rnf4 and the DNA damage response (DDR) in vivo, we generated an Rnf4 allelic series in mice. We show that Rnf4-deficiency causes persistent ionizing radiation-induced DNA damage and signaling, and that Rnf4-deficient cells and mice exhibit increased sensitivity to genotoxic stress. Mechanistically, we show that Rnf4 targets SUMOylated MDC1 and SUMOylated BRCA1, and is required for the loading of Rad51, an enzyme required for HR repair, onto sites of DNA damage. Similarly to inactivating mutations in other key regulators of HR repair, Rnf4 deficiency leads to age-dependent impairment in spermatogenesis. These findings identify Rnf4 as a critical component of the DDR in vivo and support the possibility that Rnf4 controls protein localization at DNA damage sites by integrating SUMOylation and ubiquitylation events.

Loss of PPP2R2A Inhibits Homologous Recombination DNA Repair and Predicts Tumor Sensitivity to PARP Inhibition

Reversible phosphorylation plays a critical role in DNA repair. Here, we report the results of a loss-of-function screen that identifies the PP2A heterotrimeric serine/threonine phosphatases PPP2R2A, PPP2R2D, PPP2R5A, and PPP2R3C in double-strand break (DSB) repair. In particular, we found that PPP2R2A-containing complexes directly dephosphorylated ATM at S367, S1893, and S1981 to regulate its retention at DSB sites. Increased ATM phosphorylation triggered by PPP2R2A attenuation dramatically upregulated the activity of the downstream effector kinase CHK2, resulting in G(1) to S-phase cell-cycle arrest and downregulation of BRCA1 and RAD51. In tumor cells, blocking PPP2R2A thereby impaired the high-fidelity homologous recombination repair pathway and sensitized cells to small-molecule inhibitors of PARP. We found that PPP2R2A was commonly downregulated in non-small cell lung carcinomas, suggesting that PPP2R2A status may serve as a marker to predict therapeutic efficacy to PARP inhibition. In summary, our results deepen understanding of the role of PP2A family phosphatases in DNA repair and suggest PPP2R2A as a marker for PARP inhibitor responses in clinic.

p16INK4a Impairs Homologous Recombination–Mediated DNA Repair in Human Papillomavirus–Positive Head and Neck Tumors

The p16INK4a protein is a principal cyclin-dependent kinase inhibitor that decelerates the cell cycle. Abnormally high levels of p16INK4a are commonly observed in human papillomavirus (HPV)-positive head and neck squamous cell carcinomas (HNSCC). We and others found that p16INK4a overexpression is associated with improved therapy response and survival of patients with HNSCC treated with radiotherapy. However, the functional role of p16INK4a in HNSCC remains unexplored. Our results implicate p16INK4a in regulation of homologous recombination-mediated DNA damage response independently from its role in control of the cell cycle. We found that expression of p16INK4a dramatically affects radiation sensitivity of HNSCC cells. p16INK4a overexpression impairs the recruitment of RAD51 to the site of DNA damage in HPV-positive cells by downregulating of cyclin D1 protein expression. Consistent with the in vitro findings, immunostaining of HNSCC patient samples revealed that high levels p16INK4a expression significantly correlated with decreased cyclin D1 expression. In summary, these findings reveal an unexpected function of p16INK4a in homologous recombination-mediated DNA repair response and imply p16INK4a status as an independent marker to predict response of patients with HNSCC to radiotherapy.

Loss of Chromosome 8p Governs Tumor Progression and Drug Response by Altering Lipid Metabolism

Large-scale heterozygous deletions are a hallmark of cancer genomes. The concomitant loss of multiple genes creates vulnerabilities that are impossible to reveal through the study of individual genes. To delineate the functional outcome of chromosome 8p loss of heterozygosity (LOH), a common aberration in breast cancer, we modeled 8p LOH using TALEN-based genomic engineering. 8p LOH alters fatty acid and ceramide metabolism. The shift in lipid metabolism triggers invasiveness and confers tumor growth under stress conditions due to increased autophagy. The resistance of 8p-deleted cells to chemotherapeutic drugs concurs with poorer survival rates of breast cancer patients harboring an 8p LOH. The autophagy dependency of 8p-deleted cells provides the rational basis for treatment of 8p LOH tumors with autophagy inhibitors.

OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination

Activation of the RAS oncogenic pathway, frequently ensuing from mutations in RAS genes, is a common event in human cancer. Recent reports demonstrate that reversible ubiquitination of RAS GTPases dramatically affects their activity, suggesting that enzymes involved in regulating RAS ubiquitination may contribute to malignant transformation. Here, we identified the de‐ubiquitinase OTUB1 as a negative regulator of RAS mono‐ and di‐ubiquitination. OTUB1 inhibits RAS ubiquitination independently of its catalytic activity resulting in sequestration of RAS on the plasma membrane. OTUB1 promotes RAS activation and tumorigenesis in wild‐type RAS cells. An increase of OTUB1 expression is commonly observed in non‐small‐cell lung carcinomas harboring wild‐type KRAS and is associated with increased levels of ERK1/2 phosphorylation, high Ki67 score, and poorer patient survival. Our results strongly indicate that dysregulation of RAS ubiquitination represents an alternative mechanism of RAS activation during lung cancer development.

PP2A Inactivation Mediated by PPP2R4 Haploinsufficiency Promotes Cancer Development

Protein phosphatase 2A (PP2A) complexes counteract many oncogenic kinase pathways. In cancer cells, PP2A function can be compromised by several mechanisms, including sporadic mutations in its scaffolding A and regulatory B subunits or more frequently through overexpression of cellular PP2A inhibitors. Here, we identify a novel genetic mechanism by which PP2A function is recurrently affected in human cancer, involving haploinsufficiency of PPP2R4, a gene encoding the cellular PP2A activator PTPA. Notably, up to 70% of cancer patients showed a heterozygous deletion or missense mutations in PPP2R4 Cancer-associated PTPA mutants exhibited decreased abilities to bind the PP2A-C subunit or activate PP2A and failed to reverse the tumorigenic phenotype induced by PTPA suppression, indicating they function as null alleles. In Ppp2r4 gene-trapped (gt) mice showing residual PTPA expression, total PP2A activity and methylation were reduced, selectively affecting specific PP2A holoenzymes. Both PTPAgt/gt and PTPA+/gt mice showed higher rates of spontaneous tumors, mainly hematologic malignancies and hepatocellular adenomas and carcinomas. These tumors exhibited increased c-Myc phosphorylation and increased Wnt or Hedgehog signaling. We observed a significant reduction in lifespan in PTPA+/gt mice compared with wild-type mice. In addition, chemical-induced skin carcinogenesis was accelerated in PTPA+/gt compared with wild-type mice. Our results provide evidence for PPP2R4 as a haploinsufficient tumor suppressor gene, defining a high-penetrance genetic mechanism for PP2A inhibition in human cancer. Cancer Res; 77(24); 6825-37. ©2017 AACR.

Therapeutic relevance of the protein phosphatase 2A in cancer

Chromosomal Instability (CIN) is regarded as a unifying feature of heterogeneous tumor populations, driving intratumoral heterogeneity. Polo-Like Kinase 1 (PLK1), a serine-threonine kinase that is often overexpressed across multiple tumor types, is one of the key regulators of CIN and is considered as a potential therapeutic target. However, targeting PLK1 has remained a challenge due to the off-target effects caused by the inhibition of other members of the polo-like family. Here we use synthetic dosage lethality (SDL), where the overexpression of PLK1 is lethal only when another, normally non-lethal, mutation or deletion is present. Rather than directly inhibiting PLK1, we found that inhibition of PP2A causes selective lethality to PLK1-overexpressing breast, pancreatic, ovarian, glioblastoma, and prostate cancer cells. As PP2A is widely regarded as a tumor suppressor, we resorted to gene expression datasets from cancer patients to functionally dissect its therapeutic relevance. We identified two major classes of PP2A subunits that negatively correlated with each other. Interestingly, most mitotic regulators, including PLK1, exhibited SDL interactions with only one class of PP2A subunits (PPP2R1A, PPP2R2D, PPP2R3B, PPP2R5B and PPP2R5D). Validation studies and other functional cell-based assays showed that inhibition of PPP2R5D affects both levels of phospho-Rb as well as sister chromatid cohesion in PLK1-overexpressing cells. Finally, analysis of clinical data revealed that patients with high expression of mitotic regulators and low expression of Class I subunits of PP2A improved survival. Overall, these observations point to a context-dependent role of PP2A that warrants further exploration for therapeutic benefits.

Nuclear p16INK4a expression predicts enhanced radiation response in head and neck cancers

Immunohistochemistry analysis of p16INK4a in head and neck squamous cell carcinomas (HNSCC) tumor samples revealed that 28% of tumors showed nuclear/cytoplasmic p16INK4a localization, while 37% of tumors had cytoplasmic p16INK4a. Our previous study showed that p16INK4a inhibits the DNA repair response independently of its function in the cell cycle, suggesting that p16INK4a subcellular localization should be considered during stratification of HNSCC patients. Using p16INK4a mutants with different localization signals, we found that expression of nuclear p16INK4a, but not cytoplasmic p16INK4a impaired RAD51 foci formation, indicating that nuclear localization of p16INK4a is crucial for its function in DNA repair. We next investigated the role of p16INK4a subcellular localization in radiation response in a retrospective cohort of 261 HNSCC patients treated with chemoradiation. We found that only HNSCC patients expressing nuclear p16INK4a expression showed better outcome, locoregional control and disease free survival, after chemoradiation. In concordance with the patient data, only expression of nuclear p16INK4a increased radiosensitivity of HNSCC cells. These results implicate nuclear p16INK4a expression as a potent marker to predict radiation response of HNSCC patients and should be taken into account in intensification or de-escalation studies.

Abstract 1218: PTPRO as a candidate phosphatase in regulating EGFR signaling in colorectal cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background Protein tyrosine phosphatases (PTPs) are known to be responsible for the negative regulation of many receptor tyrosine kinases by serving as antagonists to tyrosine kinase signaling. Several PTPs have been identified as candidate regulators of EGFR phosphorylation such as PTP1B, RPTP-sigma, RPTP-kappa, and PTPRJ (DEP-1). Here, we focused on PTPRO, a member of R3 family. The Drosophila ortholog of this family, Ptp4E/Ptp10D, negatively regulates EGFR signalling. Gene expression analysis of 688 primary tumours led us to identify PTPRO as a gene with strongly reduced expression in colorectal cancers with poor prognosis in particular the ones belonging to the Braf mutant subgroup. This suggests that PTPRO could act as a tumour suppressor by negative regulation of EGFR in colorectal cancer. Material and methods HEK293 cells were transiently transfected with wild-type (WT) PTPRO and with its catalytic mutant (C/S) and stimulated with EGF. After cell lysis, extracts were analyzed by SDS-PAGE and probed by western blotting with an anti-phosphotyrosine antibody (pY99, 4G10). The total level of EGFR is detected with anti-EGFR antibody. EGFR phosphorylation antibody arrays are used to identify the specific tyrosine phosphorylation sites. Results and discussion Upon expression of the PTPRO (C/S) mutant, we observe a delay in EGF induced EGFR degradation. Our preliminary data suggests that PTPRO might play a role in EGFR trafficking through the endocytic pathway and its catalytic activity is required for efficient endocytic progression of EGFR. Ongoing experiments will answer to this question. WT-PTPRO over-expression specifically decreases the Tyr 845 phosphorylation of EGFR, a known Src family tyrosine kinase (SFK) phosphorylation site. SFKs also regulate receptor turnover both at the level of endocytosis and cbl-mediated ubiquitination by phosphorylation. However, underlying mechanism how PTPRO can modulate EGFR signalling through the regulation of endocytic machinery is not known yet. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1218. doi:1538-7445.AM2012-1218