Brittany Haynes

The Escherichia coli CydX Protein Is a Member of the CydAB Cytochrome bd Oxidase Complex and Is Required for Cytochrome bd Oxidase Activity

Cytochrome bd oxidase operons from more than 50 species of bacteria contain a short gene encoding a small protein that ranges from ∼30 to 50 amino acids and is predicted to localize to the cell membrane. Although cytochrome bd oxidases have been studied for more than 70 years, little is known about the role of this small protein, denoted CydX, in oxidase activity. Here we report that Escherichia coli mutants lacking CydX exhibit phenotypes associated with reduced oxidase activity. In addition, cell membrane extracts from ΔcydX mutant strains have reduced oxidase activity in vitro. Consistent with data showing that CydX is required for cytochrome bd oxidase activity, copurification experiments indicate that CydX interacts with the CydAB cytochrome bd oxidase complex. Together, these data support the hypothesis that CydX is a subunit of the CydAB cytochrome bd oxidase complex that is required for complex activity. The results of mutation analysis of CydX suggest that few individual amino acids in the small protein are essential for function, at least in the context of protein overexpression. In addition, the results of analysis of the paralogous small transmembrane protein AppX show that the two proteins could have some overlapping functionality in the cell and that both have the potential to interact with the CydAB complex.

Crosstalk between translesion synthesis, Fanconi anemia network, and homologous recombination repair pathways in interstrand DNA crosslink repair and development of chemoresistance.

Bifunctional alkylating and platinum based drugs are chemotherapeutic agents used to treat cancer. These agents induce DNA adducts via formation of intrastrand or interstrand (ICL) DNA crosslinks, and DNA lesions of the ICL type are particularly toxic as they block DNA replication and/or DNA transcription. However, the therapeutic efficacies of these drugs are frequently limited due to the cancer cells enhanced ability to repair and tolerate these toxic DNA lesions. This ability to tolerate and survive the DNA damage is accomplished by a set of specialized low fidelity DNA polymerases called translesion synthesis (TLS) polymerases since high fidelity DNA polymerases are unable to replicate the damaged DNA template. TLS is a crucial initial step in ICL repair as it synthesizes DNA across the lesion thus preparing the damaged DNA template for repair by the homologous recombination (HR) pathway and Fanconi anemia (FA) network, processes critical for ICL repair. Here we review the molecular features and functional roles of TLS polymerases, discuss the collaborative interactions and cross-regulation of the TLS DNA damage tolerance pathway, the FA network and the BRCA-dependent HRR pathway, and the impact of TLS hyperactivation on development of chemoresistance. Finally, since TLS hyperactivation results from overexpression of Rad6/Rad18 ubiquitinating enzymes (fundamental components of the TLS pathway), increased PCNA ubiquitination, and/or increased recruitment of TLS polymerases, the potential benefits of selectively targeting critical components of the TLS pathway for enhancing anti-cancer therapeutic efficacy and curtailing chemotherapy-induced mutagenesis are also discussed.

Gold nanoparticle conjugated Rad6 inhibitor induces cell death in triple negative breast cancer cells by inducing mitochondrial dysfunction and PARP-1 hyperactivation: Synthesis and characterization.

UNLABELLED We recently developed a small molecule inhibitor SMI#9 for Rad6, a protein overexpressed in aggressive breast cancers and involved in DNA damage tolerance. SMI#9 induces cytotoxicity in cancerous cells but spares normal breast cells; however, its therapeutic efficacy is limited by poor solubility. Here we chemically modified SMI#9 to enable its conjugation and hydrolysis from gold nanoparticle (GNP). SMI#9-GNP and parent SMI#9 activities were compared in mesenchymal and basal triple negative breast cancer (TNBC) subtype cells. Whereas SMI#9 is cytotoxic to all TNBC cells, SMI#9-GNP is endocytosed and cytotoxic only in mesenchymal TNBC cells. SMI#9-GNP endocytosis in basal TNBCs is compromised by aggregation. However, when combined with cisplatin, SMI#9-GNP is imported and synergistically increases cisplatin sensitivity. Like SMI#9, SMI#9-GNP spares normal breast cells. The released SMI#9 is active and induces cell death via mitochondrial dysfunction and PARP-1 stabilization/hyperactivation. This work signifies the development of a nanotechnology-based Rad6-targeting therapy for TNBCs. FROM THE CLINICAL EDITOR Protein Rad6 is overexpressed in breast cancer cells and its blockade may provide a new treatment against 3N breast cancer. The authors conjugated a small molecule inhibitor SMI#9 for Rad6 to gold nanoparticles in this study and showed that this new formulation specifically targeted chemo-resistant breast cancer cells and highlighted the importance of nanotechnology in drug carrier development.

Melanoma Development and Progression Are Associated with Rad6 Upregulation and β-Catenin Relocation to the Cell Membrane

We have previously demonstrated that Rad6 and β-catenin enhance each others expression through a positive feedback loop to promote breast cancer development/progression. While β-catenin has been implicated in melanoma pathogenesis, Rad6 function has not been investigated. Here, we examined the relationship between Rad6 and β-catenin in melanoma development and progression. Eighty-eight cutaneous tumors, 30 nevi, 29 primary melanoma, and 29 metastatic melanomas, were immunostained with anti-β-catenin and anti-Rad6 antibodies. Strong expression of Rad6 was observed in only 27% of nevi as compared to 100% of primary and 96% of metastatic melanomas. β-Catenin was strongly expressed in 97% of primary and 93% of metastatic melanomas, and unlike Rad6, in 93% of nevi. None of the tumors expressed nuclear β-catenin. β-Catenin was exclusively localized on the cell membrane of 55% of primary, 62% of metastatic melanomas, and only 10% of nevi. Cytoplasmic β-catenin was detected in 90% of nevi, 17% of primary, and 8% of metastatic melanoma, whereas 28% of primary and 30% of metastatic melanomas exhibited β-catenin at both locations. These data suggest that melanoma development and progression are associated with Rad6 upregulation and membranous redistribution of β-catenin and that β-catenin and Rad6 play independent roles in melanoma development.

Rad6 is a Potential Early Marker of Melanoma Development

Melanoma is the leading cause of death from skin cancer in industrialized countries. Several melanoma-related biomarkers and signaling pathways have been identified; however, their relevance to melanoma development/progression or to clinical outcome remains to be established. Aberrant activation of Wnt/β-catenin pathway is implicated in various cancers including melanoma. We have previously demonstrated Rad6, an ubiquitin-conjugating enzyme, as an important mediator of β-catenin stability in breast cancer cells. Similar to breast cancer, β-catenin-activating mutations are rare in melanomas, and since β-catenin signaling is implicated in melanoma, we examined the relationship between β-catenin levels/activity and expression of β-catenin transcriptional targets Rad6 and microphthalmia-associated transcription factor-M (Mitf-M) in melanoma cell models, and expression of Rad6, β-catenin, and Melan-A in nevi and cutaneous melanoma tissue specimens. Our data show that Rad6 is only weakly expressed in normal human melanocytes but is overexpressed in melanoma lines. Unlike Mitf-M, Rad6 overexpression in melanoma lines is positively associated with high molecular weight β-catenin protein levels and β-catenin transcriptional activity. Double-immunofluorescence staining of Rad6 and Melan-A in melanoma tissue microarray showed that histological diagnosis of melanoma is significantly associated with Rad6/Melan-A dual positivity in the melanoma group compared to the nevi group (P = .0029). In contrast to strong β-catenin expression in normal and tumor areas of superficial spreading malignant melanoma (SSMM), Rad6 expression is undetectable in normal areas and Rad6 expression increases coincide with increased Melan-A in the transformed regions of SSMM. These data suggest a role for Rad6 in melanoma pathogenesis and that Rad6 expression status may serve as an early marker for melanoma development.

Breast cancer complexity: implications of intratumoral heterogeneity in clinical management

Generation of intratumoral phenotypic and genetic heterogeneity has been attributed to clonal evolution and cancer stem cells that together give rise to a tumor with complex ecosystems. Each ecosystem contains various tumor cell subpopulations and stromal entities, which, depending upon their composition, can influence survival, therapy responses, and global growth of the tumor. Despite recent advances in breast cancer management, the disease has not been completely eradicated as tumors recur despite initial response to treatment. In this review, using data from clinically relevant breast cancer models, we show that the fates of tumor stem cells/progenitor cells in the individual tumor ecosystems comprising a tumor are predetermined to follow a limited (unipotent) and/or unlimited (multipotent) path of differentiation which create conditions for active generation and maintenance of heterogeneity. The resultant dynamic systems respond differently to treatments, thus disrupting the delicate stability maintained in the heterogeneous tumor. This raises the question whether it is better then to preserve stability by preventing takeover by otherwise dormant ecosystems in the tumor following therapy. The ultimate strategy for personalized therapy would require serial assessments of the patient’s tumor for biomarker validation during the entire course of treatment that is combined with their three-dimensional mapping to the tumor architecture and landscape.

Pharmacological targeting of Rad6 enzyme-mediated translesion synthesis overcomes resistance to platinum-based drugs

Platinum drug-induced cross-link repair requires the concerted activities of translesion synthesis (TLS), Fanconi anemia (FA), and homologous recombination repair pathways. The E2 ubiquitin-conjugating enzyme RAD6 is essential for TLS. Here, we show that RAD6 plays a universal role in platinum-based drug tolerance. Using a novel RAD6-selective small-molecule inhibitor (SMI#9) targeting the RAD6 catalytic site, we demonstrate that SMI#9 potentiates the sensitivities of cancer cells with innate or acquired cisplatin or oxaliplatin resistance. 5-Iododeoxyuridine/5-chlorodeoxyuridine pulse-labeling experiments showed that RAD6 is necessary for overcoming cisplatin-induced replication fork stalling, as replication-restart was impaired in both SMI#9-pretreated and RAD6B-silenced cells. Consistent with the role of RAD6/TLS in late-S phase, SMI#9-induced DNA replication inhibition occurred preferentially in mid/late-S phase. The compromised DNA repair and chemosensitization induced by SMI#9 or RAD6B depletion were associated with decreased platinum drug-induced proliferating cell nuclear antigen (PCNA) and FANCD2 monoubiquitinations (surrogate markers of TLS and FA pathway activation, respectively) and with attenuated FANCD2, RAD6, γH2AX, and POL η foci formation and cisplatin-adduct removal. SMI#9 pretreatment synergistically increased cisplatin inhibition of MDA-MB-231 triple-negative breast cancer cell proliferation and tumor growth. Using an isogenic HCT116 colon cancer model of oxaliplatin resistance, we further show that γH2AX and monoubiquitinated PCNA and FANCD2 are constitutively up-regulated in oxaliplatin-resistant HCT116 (HCT116-OxR) cells and that γH2AX, PCNA, and FANCD2 monoubiquitinations are induced by oxaliplatin in parental HCT116 cells. SMI#9 pretreatment sensitized HCT116-OxR cells to oxaliplatin. These data deepen insights into the vital role of RAD6/TLS in platinum drug tolerance and reveal clinical benefits of targeting RAD6 with SMI#9 for managing chemoresistant cancers.

Abstract 3786: Rad6 inhibition enhances paclitaxel sensitivity of triple negative breast cancer cells by inducing mitotic spindle defects

Triple negative breast cancers (TNBC) lack estrogen receptors, and progesterone receptors, and HER2/neu receptor amplifications, making targeted therapies unsuitable. The taxane based drug paclitaxel (PTX) is used as a first line chemotherapy. PTX is a microtubule stabilizer that induces G2/M arrest and mitotic catastrophe. Loss of BRCA1 often seen in TNBC patients is associated with PTX resistance. Rad6 is an E2 ubiquitin conjugating enzyme that has two human homologues, Rad6A (UBE2A) and Rad6B (UBE2B), and high Rad6B expression rather than Rad6A is associated with poor survival of TNBC patients. Rad6 is associated with centrosomes at all phases of the cell cycle, and constitutive overexpression of Rad6B in nontransformed MCF10A breast cells leads to centrosome amplification and aneuploidy. Inhibition of Rad6 enzymatic activity, with our novel Rad6-selective small molecule inhibitor (SMI#9) induces G2/M arrest, much like PTX. We hypothesize that the overexpression of Rad6B commonly found in BRCA1 wt and BRCA1 mut TNBCs contributes to taxane resistance by promoting centrosome amplification and enhancing microtubule dynamics. Thus, targeting Rad6 will confer taxane sensitivity by preventing centrosome reduplication and/or microtubule hypernucleation, strengthening G2/M arrest, and ensuring terminal mitotic catastrophe. Immunostaining shows Rad6 is overexpressed in BRCA1 wt and BRCA1 mut breast cancer tissues and TNBC cell lines. Immunostaining of the centrosomal protein pericentrin shows centrosome amplification in both BRCA1 wt and BRCA1 mut TNBC cells. MTT data show SMI#9 synergistically sensitizes both BRCA1 wt (MDA-MB-468) and BRCA1 mut (HCC1937) TNBC cell lines to PTX. Colony forming assays corroborate these data and show that addition of SMI#9 diminishes colony forming efficiency of PTX pretreated MDA-MB-468 cells. SMI#9 treatment of HCC1937 PTX resistant colonies results in increases in cells with enlarged and multiple nuclei (characteristic of mitotic catastrophe). Western blot analysis of Tau, a marker of PTX sensitivity, indicates PTX, SMI#9, and PTX+SMI#9 treatments decrease the steady state levels of the 1N3R and 0N4R, or 1N3R Tau isoforms in MDA-MB-468 and HCC1937 cells, respectively. Analysis of cyclin B1, a marker of G2/M arrest, shows increases in cyclin B1 levels in PTX, SMI#9, and PTX+SMI#9 treated MDA-MB-468 cells, whereas cyclin B1 is degraded in HCC1937 cells treated with these agents (indicative of mitotic catastrophe). Immunofluorescence staining shows that Tau localization to the mitotic spindles is unaffected by PTX, SMI#9, or PTX+SMI#9. However, treatments including SMI#9 resulted in mitotic cells with defective or monopolar mitotic spindles. These data implicate a role for Rad6 in centrosome duplication/separation and provide mechanistic support for inhibiting Rad6 to enhance PTX sensitivity. Supported by NCI R21 CA178117 and T32-CA009531. Citation Format: Brittany Haynes, Kristen Cunningham, Malathy Shekhar. Rad6 inhibition enhances paclitaxel sensitivity of triple negative breast cancer cells by inducing mitotic spindle defects. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3786.

Abstract 1662: Therapeutic relevance of the Rad6/translesion synthesis pathway in BRCA1-related triple-negative breast cancer cells

Triple negative breast cancers (TNBCs) comprise ∼15-20% of all breast cancers (BrCas), and their treatment is challenging because they are not treatable with ER, PgR or Her2/neu-targeted therapies. Since TNBCs and BRCA-mutated BrCas share many histopathologic features including aberrant DNA repair pathways, the concept of targeting DNA repair defects with agents such as platinum (Pt) compounds is applied for treatment of TNBC patients. Pt drugs induce DNA replication stalling interstrand DNA crosslinks (ICLs), the repair of which require concerted activities of nucleotide excision repair, Fanconi anemia (FA)/BRCA homologous recombination repair (HRR) and translesion synthesis (TLS) pathways. The therapeutic efficacies of these drugs are often limited by the cancer cell9s enhanced ability to repair/tolerate these toxic DNA lesions. The TLS pathway a.k.a DNA damage tolerance pathway is critical for cell survival in the face of DNA damage. TLS is a crucial initial step in ICL repair as it synthesizes DNA across the lesion thus preparing the damaged DNA template for repair by the FA network and HR pathway, processes critical for ICL repair. The Rad6 gene is a principal component of the TLS pathway and previous work in our lab has shown its important role in BrCa development/progression and acquisition of cisplatin (CDDP) resistance. To understand the involvements of Rad6/TLS and FA/BRCA in CDDP-induced DNA damage repair, we evaluated CDDP sensitivities, CDDP-induced DNA damage responses and stalled replication restarts in wt-BRCA1 (MDA-MB-231, MDA-MB-468) and mut-BRCA1 (HCC1937, SUM1315) TNBCs. Data from MTT assays showed no correlation between CDDP sensitivity and BRCA1 status; however, pretreatment with our recently developed Rad6 small molecule inhibitor (SMI) enhanced CDDP sensitivity of all TNBC cell lines regardless of their BRCA1 status. Consistent with these data, immunoblot analysis showed that treatment with the Rad6 SMI attenuated the CDDP-induced PCNA ubiquitinations (a hall mark of Rad6/TLS activity), as well as the steady-state levels of FancD2 (a surrogate marker of FA pathway activation), pol eta (TLS polymerase), and Rad51 (critical for HRR) in both wt-BRCA1 and mut-BRCA1 TNBC cells. IdU/CldU labeling assays showed that Rad6 inhibition mitigated the restart of CDDP-induced stalled replication forks. Whereas Rad6 is expressed weakly in normal breast tissues and overexpressed in BrCas, immunohistochemical analysis showed strong nuclear staining for Rad6 in the normal ducts of mut-BRCA1 clinical breast tissues implicating an important role for Rad6 in mut-BRCA1 BrCas. Our data suggest an important role for the Rad6/TLS pathway in processing Pt-induced ICLs in both wt-BRCA1 and mut-BRCA1 TNBCs and the potential therapeutic value of inhibiting this pathway in TNBCs. Supported by NIH CA178117-01. Note: This abstract was not presented at the meeting. Citation Format: Brittany Haynes, Hend Kothayer, Andrew Westwell, Malathy Shekhar. Therapeutic relevance of the Rad6/translesion synthesis pathway in BRCA1-related triple-negative breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1662. doi:10.1158/1538-7445.AM2015-1662

Abstract 835: COPD and risk of lung cancer in post-menopausal women: findings from the Women's Health Initiative

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Lung cancer and chronic obstructive pulmonary disease (COPD) are leading causes of morbidity and mortality in the United States. Cigarette smoking is associated with both lung cancer and COPD, but whether COPD independently predicts lung cancer risk is debated. To examine this question in a large, prospective study, we constructed risk models using data from the observational study arm of the Womens Health Initiative. Methods: Initial analyses included 89,384 women (n = 1,479 lung cancers) with complete baseline information on cigarette smoking and COPD status. We created a second, time-varying model to account for incident smoking that occurred during the eight year follow up period, in addition to the number of pack years reported at baseline. Estimates from Cox proportional hazard models were used to assess the association between COPD and lung cancer. The baseline hazard was stratified by age at study entry in all models. COPD status was self-reported, and diagnoses within one year of lung cancer diagnoses were not included. Reported lung cancers were adjudicated via medical record. Results: There was an interaction between number of pack years (continuous) of smoking at baseline and COPD status (p<0.001). Never smokers with COPD were 2.7 times more likely to develop lung cancer compared to never smokers without COPD, after adjusting for race, education, income and body mass index (HR = 2.70, 95% CI: 2.12, 3.45). Similar risk of lung cancer associated with COPD was seen among smokers with 20 pack years of smoking (HR = 2.37, 95% CI: 1.96, 2.87) and at 40 pack years of smoking (HR = 2.08, 95% CI: 1.77, 2.45) after adjustment. From the time-varying models, women with COPD had approximately 2 times the risk of developing lung cancer compared to those who did not, after adjusting for smoking, race, education, income and body mass index (HR = 1.90, 95% CI: 1.54, 2.34). COPD was associated with all subtypes of lung cancer after adjustment for smoking, with hazard ratios ranging from 1.59-2.15 for different histological subtypes of non-small cell lung cancers. Smokers with COPD were 4.6 times more likely to develop small cell lung cancers compared to those without COPD (HR = 4.68, 95% CI: 2.67, 8.20). Conclusion: These data suggest that COPD is an independent risk factor for the development of lung cancer, and should be considered when constructing risk models. Citation Format: Michele L. Cote, Amy Lehman, Rowen Chlebowski, Brittany Haynes, Gloria Ho, Manali Patel, Lori Sakoda, Michael Simon, Heather Wakelee, Ann Schwartz. COPD and risk of lung cancer in post-menopausal women: findings from the Womens Health Initiative. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 835. doi:10.1158/1538-7445.AM2015-835