David Dornan

The ubiquitin ligase COP1 is a critical negative regulator of p53

COP1 (constitutively photomorphogenic 1) is a RING-finger-containing protein that functions to repress plant photomorphogenesis, the light-mediated programme of plant development. Mutants of COP1 are constitutively photomorphogenic, and this has been attributed to their inability to negatively regulate the proteins LAF1 (ref. 1) and HY5 (ref. 2). The role of COP1 in mammalian cells is less well characterized. Here we identify the tumour-suppressor protein p53 as a COP1-interacting protein. COP1 increases p53 turnover by targeting it for degradation by the proteasome in a ubiquitin-dependent fashion, independently of MDM2 or Pirh2, which are known to interact with and negatively regulate p53. Moreover, COP1 serves as an E3 ubiquitin ligase for p53 in vitro and in vivo, and inhibits p53-dependent transcription and apoptosis. Depletion of COP1 by short interfering RNA (siRNA) stabilizes p53 and arrests cells in the G1 phase of the cell cycle. Furthermore, we identify COP1 as a p53-inducible gene, and show that the depletion of COP1 and MDM2 by siRNA cooperatively sensitizes U2-OS cells to ionizing-radiation-induced cell death. Overall, these results indicate that COP1 is a critical negative regulator of p53 and represents a new pathway for maintaining p53 at low levels in unstressed cells.

Deubiquitinase USP9X stabilizes MCL1 and promotes tumour cell survival

MCL1 is essential for the survival of stem and progenitor cells of multiple lineages, and is unique among pro-survival BCL2 family members in that it is rapidly turned over through the action of ubiquitin ligases. B- and mantle-cell lymphomas, chronic myeloid leukaemia, and multiple myeloma, however, express abnormally high levels of MCL1, contributing to chemoresistance and disease relapse. The mechanism of MCL1 overexpression in cancer is not well understood. Here we show that the deubiquitinase USP9X stabilizes MCL1 and thereby promotes cell survival. USP9X binds MCL1 and removes the Lys 48-linked polyubiquitin chains that normally mark MCL1 for proteasomal degradation. Increased USP9X expression correlates with increased MCL1 protein in human follicular lymphomas and diffuse large B-cell lymphomas. Moreover, patients with multiple myeloma overexpressing USP9X have a poor prognosis. Knockdown of USP9X increases MCL1 polyubiquitination, which enhances MCL1 turnover and cell killing by the BH3 mimetic ABT-737. These results identify USP9X as a prognostic and therapeutic target, and they show that deubiquitinases may stabilize labile oncoproteins in human malignancies.

TRPS1 Targeting by miR-221/222 Promotes the Epithelial-to-Mesenchymal Transition in Breast Cancer

The microRNAs miR-221 and miR-222 promote a phenotype associated with metastasis and are found in a clinically aggressive form of breast cancer. Parsing Breast Cancer Subtype with MicroRNAs MicroRNAs (miRNAs), short noncoding RNAs that bind to and silence target mRNAs, have emerged as playing crucial regulatory roles not only in normal cellular processes but also in pathological conditions, such as cancer. Stinson et al. analyzed miRNA expression in different types of human breast cancer and found that miR-221 and miR-222 (miR-221/222) abundance was increased in the clinically aggressive basal-like subtype compared to the less aggressive luminal subtype. They determined that signaling through the epidermal growth factor receptor (EGFR)–RAS–extracellular signal–regulated kinase (ERK) pathway increased miR-221/222 transcription, and they defined a transcriptional regulatory pathway through which miR-221/222 promoted a phenotype associated with cancer cell invasion and metastasis. Their data suggest that combining inhibition of the EGFR-RAS-ERK pathway with standard chemotherapy could, by limiting miR-221/222 production, provide a strategy to combat metastasis in the basal-like subtype of breast cancer. The basal-like subtype of breast cancer has an aggressive clinical behavior compared to that of the luminal subtype. We identified the microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) as basal-like subtype–specific miRNAs and showed that expression of miR-221/222 decreased expression of epithelial-specific genes and increased expression of mesenchymal-specific genes, and increased cell migration and invasion in a manner characteristic of the epithelial-to-mesenchymal transition (EMT). The transcription factor FOSL1 (also known as Fra-1), which is found in basal-like breast cancers but not in the luminal subtype, stimulated the transcription of miR-221/222, and the abundance of these miRNAs decreased with inhibition of the epidermal growth factor receptor (EGFR) or MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase), placing miR-221/222 downstream of the RAS pathway. Furthermore, miR-221/222–mediated reduction in E-cadherin abundance depended on their targeting the 3′ untranslated region of the GATA family transcriptional repressor TRPS1 (tricho-rhino-phalangeal syndrome type 1), which inhibited EMT by decreasing ZEB2 (zinc finger E-box–binding homeobox2) expression. We conclude that by promoting EMT, miR-221/222 may contribute to the more aggressive clinical behavior of basal-like breast cancers.

ATM Engages Autodegradation of the E3 Ubiquitin Ligase COP1 After DNA Damage

The ataxia telangiectasia mutated (ATM) protein kinase is a critical component of a DNA-damage response network configured to maintain genomic integrity. The abundance of an essential downstream effecter of this pathway, the tumor suppressor protein p53, is tightly regulated by controlled degradation through COP1 and other E3 ubiquitin ligases, such as MDM2 and Pirh2; however, the signal transduction pathway that regulates the COP1-p53 axis following DNA damage remains enigmatic. We observed that in response to DNA damage, ATM phosphorylated COP1 on Ser387 and stimulated a rapid autodegradation mechanism. Ionizing radiation triggered an ATM-dependent movement of COP1 from the nucleus to the cytoplasm, and ATM-dependent phosphorylation of COP1 on Ser387 was both necessary and sufficient to disrupt the COP1-p53 complex and subsequently to abrogate the ubiquitination and degradation of p53. Furthermore, phosphorylation of COP1 on Ser387 was required to permit p53 to become stabilized and to exert its tumor suppressor properties in response to DNA damage.

COP1, the Negative Regulator of p53, Is Overexpressed in Breast and Ovarian Adenocarcinomas

The tumor suppressor protein p53 plays a central role in protecting normal cells from undergoing transformation. Thus, it is fitting that cancer cells selectively dampen the p53 response to gain a selective growth advantage. In fact, the p53 gene is the most commonly mutated tumor suppressor gene in human cancers, and if the gene is not mutated, then other components of the p53 pathways are skewed to dampen the p53 response to stress. We recently identified COP1 as a novel and critical negative regulator of p53. COP1 is a RING finger-containing protein that targets p53 for degradation to the proteasome and is necessary for p53 turnover in normal and cancer cells. However, the association between COP1 and cancer remains to be determined. We performed expression analysis of COP1 in ovarian and breast cancer tissue microarrays. COP1 is significantly overexpressed in 81% (25 of 32) of breast and 44% (76 of 171) of ovarian adenocarcinoma as assessed by in situ hybridization and immunohistochemistry. Overexpression of COP1 correlated with a striking decrease in steady state p53 protein levels and attenuation of the downstream target gene, p21, in cancers that retain a wild-type p53 gene status. Overall, these results suggest that overexpression of COP1 contributes to the accelerated degradation of p53 protein in cancers and attenuates the tumor suppressor function of p53.

Therapeutic potential of an anti-CD79b antibody–drug conjugate, anti–CD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma

Here we describe the generation of an antibody-drug conjugate (ADC) consisting of a humanized anti-CD79b antibody that is conjugated to monomethylauristatin E (MMAE) through engineered cysteines (THIOMABs) by a protease cleavable linker. By using flow cytometry, we detected the surface expression of CD79b in almost all non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia patients, suggesting that anti-CD79b-vcMMAE could be widely used in these malignancies. By using NHL cell lines to simulate a patient population we discovered that a minimal cell-surface expression level of CD79b was required for in vitro activity. Within the subpopulation of cell lines above this minimal threshold, we found that sensitivity to free MMAE, mutation of cancer genes, and cell doubling time were poorly correlated with in vitro activity; however, the expression level of BCL-XL was correlated with reduced sensitivity to anti-CD79b-vcMMAE. This observation was supported by in vivo data showing that a Bcl-2 family inhibitor, ABT-263, strikingly enhanced the activity of anti-CD79b-vcMMAE. Furthermore, anti-CD79b-vcMMAE was significantly more effective than a standard-of-care regimen, R-CHOP (ie, rituximab with a single intravenous injection of 30 mg/kg cyclophosphamide, 2.475 mg/kg doxorubicin, 0.375 mg/kg vincristine, and oral dosing of 0.15 mg/kg prednisone once a day for 5 days), in 3 xenograft models of NHL. Together, these data suggest that anti-CD79b-vcMMAE could be broadly efficacious for the treatment of NHL.

miR-221/222 Targeting of Trichorhinophalangeal 1 (TRPS1) Promotes Epithelial-to-Mesenchymal Transition in Breast Cancer

MicroRNAs miR-221 and miR-222 are associated with a clinically aggressive form of breast cancer and promote epithelial-to-mesenchymal transition. Compared with the luminal subtype, the basal-like subtype of breast cancer has an aggressive clinical behavior, but the reasons for this difference between the two subtypes are poorly understood. We identified microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) as basal-like subtype-specific miRNAs that decrease expression of epithelial-specific genes and increase expression of mesenchymal-specific genes. In addition, expression of these miRNAs increased cell migration and invasion, which collectively are characteristics of the epithelial-to-mesenchymal transition (EMT). The basal-like transcription factor FOSL1 (also known as Fra-1) directly stimulated the transcription of miR-221/222, and the abundance of these miRNAs decreased with inhibition of MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase), placing miR-221/222 downstream of the RAS pathway. The miR-221/222–mediated reduction in E-cadherin abundance depended on their targeting of the 3′ untranslated region (3′UTR) of TRPS1 (trichorhinophalangeal syndrome type 1), which is a member of the GATA family of transcriptional repressors. TRPS1 inhibited EMT by directly repressing expression of ZEB2 (Zinc finger E-box–binding homeobox 2). Therefore, miR-221/222 may contribute to the aggressive clinical behavior of basal-like breast cancers.

The Proline Repeat Domain of p53 Binds Directly to the Transcriptional Coactivator p300 and Allosterically Controls DNA-Dependent Acetylation of p53

ABSTRACT The transcription coactivator p300 cannot acetylate native p53 tetramers, thus revealing intrinsic conformational constraints on p300-catalyzed acetylation. Consensus site DNA is an allosteric effector that promotes acetylation of p53, suggesting that p300 has an undefined conformationally flexible interface within the p53 tetramer. To identify such conformationally responsive p300-binding sites, p300 was subjected to peptide selection from a phage-peptide display library, a technique that can define novel protein-protein interfaces. The enriched p300-binding peptides contained a proline repeat (PXXP/PXPXP) motif, and five proline repeat motifs actually reside within the p53 transactivation domain, suggesting that this region of p53 may harbor the second p300 contact site. p300 binds in vitro to PXXP-containing peptides derived from the proline repeat domain, and PXXP-containing peptides inhibit sequence-specific DNA-dependent acetylation of p53, indicating that p300 docking to both the LXXLL and contiguous PXXP motif in p53 is required for p53 acetylation. Deletion of the proline repeat motif of p53 prevents DNA-dependent acetylation of p53 by occluding p300 from the p53-DNA complex. Sequence-specific DNA places an absolute requirement for the proline repeat domain to drive p53 acetylation in vivo. Chromatin immunoprecipitation was used to show that the proline repeat deletion mutant p53 is bound to the p21 promoter in vivo, but it is not acetylated, indicating that proline-directed acetylation of p53 is a post-DNA binding event. The PXXP repeat expands the basic interface of a p300-targeted transactivation domain, and proline-directed acetylation of p53 at promoters indicates that p300-mediated acetylation can be highly constrained by substrate conformation in vivo.

miR-221/222 Targets Adiponectin Receptor 1 to Promote the Epithelial-to-Mesenchymal Transition in Breast Cancer

The epithelial-to-mesenchymal transition (EMT) is a highly conserved physiological program involved in development and tissue repair; however, its aberrant activation has been implicated in accelerating the progression of a variety of cancers. In breast cancer, the microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) are differentially expressed in the clinically more aggressive basal-like subtype compared to luminal subtype of breast cancer and upregulation of miR-221/222 induces the EMT by targeting the 3′ untranslated region (3′UTR) of the GATA family transcriptional repressor TRPS1 (tricho-rhino-phalangeal syndrome type 1). The complete mechanism through which miR-221/222 promotes the EMT, however, is not fully understood. We identified adiponectin receptor 1 (ADIPOR1), a receptor for the adipocytokine adiponectin, as a direct target of miR-221/222. ADIPOR1 is expressed at higher levels in the luminal compared to the basal-like subtype of breast cancer cell lines, which can be reduced by miR-221/222 targeting of its 3’UTR. In addition, miR-221/222 were negatively correlated with ADIPOR1 expression across breast cancer cell lines and tumors. ADIPOR1 depletion by siRNA in MCF10A cells induced the EMT and increased cell invasion. Depletion of ADIPOR1 by siRNA induced activation of the canonical nuclear factor-kappaB (NF-κB) and subsequent phosphorylation of signal transducer and activator of transcription 3 (STAT3) in an interleukin 6 (IL6)-dependent manner. Finally, overexpression of ADIPOR1 in the basal-like cell line, MDA-MB-231, attenuated cell invasion and promoted the mesenchymal-to-epithelial transition (MET). We conclude that ADIPOR1 negatively regulates EMT in breast cancer and provides an additional node by which miR-221/222 induces the EMT. These results suggest that ADIPOR1 may play an important role in breast cancer progression and metastasis, and could potentially offer an alternative therapeutic strategy for basal-like breast cancer patients.

DCDT2980S, an Anti-CD22-Monomethyl Auristatin E Antibody–Drug Conjugate, Is a Potential Treatment for Non-Hodgkin Lymphoma

Antibody–drug conjugates (ADC), potent cytotoxic drugs linked to antibodies via chemical linkers, allow specific targeting of drugs to neoplastic cells. We have used this technology to develop the ADC DCDT2980S that targets CD22, an antigen with expression limited to B cells and the vast majority of non-Hodgkin lymphomas (NHL). DCDT2980S consists of a humanized anti-CD22 monoclonal IgG1 antibody with a potent microtubule-disrupting agent, monomethyl auristatin E (MMAE), linked to the reduced cysteines of the antibody via a protease cleavable linker, maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB). We describe the efficacy, safety, and pharmacokinetics of DCDT2980S in animal models to assess its potential as a therapeutic for the treatment of B-cell malignancies. We did not find a strong correlation between in vitro or in vivo efficacy and CD22 surface expression, nor a correlation of sensitivity to free drug and in vitro potency. We show that DCDT2980S was capable of inducing complete tumor regression in xenograft mouse models of NHL and can be more effective than rituximab plus combination chemotherapy at drug exposures that were well tolerated in cynomolgus monkeys. These results suggest that DCDT2980S has an efficacy, safety, and pharmacokinetics profile that support potential treatment of NHL. Mol Cancer Ther; 12(7); 1255–65. ©2013 AACR.