Enhanced sensitivity to cisplatin and gemcitabine in Brca1-deficient murine mammary epithelial cells.

BACKGROUND: Breast cancers due to germline mutations or altered expression of the BRCA1 gene associate with an aggressive clinical course and frequently exhibit a "triple-negative" phenotype, i.e. lack of expression of the estrogen and progesterone hormone receptors and lack of overexpression of the HER2/NEU oncogene, thereby rendering them relatively insensitive to hormonal manipulation and targeted HER2 therapy, respectively. BRCA1 plays a role in multiple DNA repair pathways, and thus, when mutated, results in sensitivity to certain DNA damaging drugs. RESULTS: Here, we used a Brca1 murine mammary epithelial cell (MMEC) model to examine the effect of loss of Brca1 on cellular sensitivity to various chemotherapy drugs. To explore novel therapeutic strategies, we included DNA damaging and non-DNA damaging drugs whose mechanisms are dependent and independent of DNA repair, respectively, and drugs that are used in standard and non-standard lines of therapy for breast cancer. To understand the cellular mechanism, we also determined the role that DNA repair plays in sensitivity to these drugs. We found that cisplatin and gemcitabine had the greatest specific therapeutic benefit to Brca1-deficient MMECs, and that when used in combination produced a synergistic effect. This sensitivity may be attributed in part to defective NER, which is one of the DNA repair pathways normally responsible for repairing DNA adducts produced by cisplatin and is shown in this study to be defective in Brca1-deficient MMECs. Brca1-deficient MMECs were not differentially sensitive to the standard breast cancer chemotherapy drugs doxorubicin, docetaxel or 5-FU. CONCLUSIONS: Both cisplatin and gemcitabine should be explored in clinical trials for first line regimens for BRCA1-associated and triple-negative breast cancer.

The role of the retinoblastoma/E2F1 tumor suppressor pathway in the lesion recognition step of nucleotide excision repair.

The retinoblastoma Rb/E2F tumor suppressor pathway plays a major role in the regulation of mammalian cell cycle progression. The pRb protein, along with closely related proteins p107 and p130, exerts its anti-proliferative effects by binding to the E2F family of transcription factors known to regulate essential genes throughout the cell cycle. We sought to investigate the role of the Rb/E2F1 pathway in the lesion recognition step of nucleotide excision repair (NER) in mouse embryonic fibroblasts (MEFs). Rb-/-, p107-/-, p130-/- MEFs repaired both cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) at higher efficiency than did wildtype cells following UV-C irradiation. The expression of damaged DNA binding gene DDB2 involved in the DNA lesion recognition step was elevated in the Rb family-deficient MEFs. To determine if the enhanced DNA repair in the absence of the Rb gene family is due to the derepression of E2F1, we assayed the ability of E2F1-deficient cells to repair damaged DNA and demonstrated that E2F1-/- MEFs are impaired for the removal of both CPDs and 6-4PPs. Furthermore, wildtype cells induced a higher expression of DDB2 and xeroderma pigmentosum gene XPC transcript levels than did E2F1-/- cells following UV-C irradiation. Using an E2F SiteScan algorithm, we uncovered a putative E2F-responsive element in the XPC promoter upstream of the transcription start site. We showed with chromatin immunoprecipitation assays the binding of E2F1 to the XPC promoter in a UV-dependent manner, suggesting that E2F1 is a transcriptional regulator of XPC. Our study identifies a novel E2F1 gene target and further supports the growing body of evidence that the Rb/E2F1 tumor suppressor pathway is involved in the regulation of the DNA lesion recognition step of nucleotide excision repair.

Disparities in Systemic Treatment Use in Advanced-stage Non-Small Cell Lung Cancer by Source of Health Insurance.

BACKGROUND: Management of advanced-stage non-small cell lung cancer (NSCLC) has changed significantly over the past two decades with the development of numerous systemic treatments, including targeted therapies. However, a high proportion of advanced-stage patients are untreated. The role that health insurance plays in receipt of systemic treatments is unclear. METHODS: Using California Cancer Registry data (2012-2014), we developed multivariable Poisson regression models to assess the independent effect of health insurance type on systemic treatment utilization among patients with stage IV NSCLC. Systemic treatment information was manually abstracted from treatment text fields. RESULTS: A total of 17,310 patients were evaluated. Patients with Medicaid/other public insurance were significantly less likely to receive any systemic treatments [risk ratio (RR), 0.78; 95% confidence interval (CI), 0.75-0.82], bevacizumab combinations (RR, 0.57; 95% CI, 0.45-0.71), or tyrosine kinase inhibitors (RR, 0.70; 95% CI, 0.60-0.82) compared with the privately insured. Patients with Medicare or dual Medicare-Medicaid insurance were not significantly different from the privately insured in their likelihood of receiving systemic treatments. CONCLUSIONS: Substantial disparities in the use of systemic treatments for stage IV NSCLC exist by source of health insurance in California. Patients with Medicaid/other public insurance were significantly less likely to receive systemic treatments compared with their privately insured counterparts. IMPACT: Source of health insurance influences care received. Further research is warranted to better understand barriers to treatment that patients with Medicaid face.

First-Line Systemic Treatments for Stage IV Non-Small Cell Lung Cancer in California: Patterns of Care and Outcomes in a Real-World Setting.

BACKGROUND: Multiple systemic treatments have been developed for stage IV non-small cell lung cancer (NSCLC), but their use and effect on outcomes at the population level are unknown. This study describes the utilization of first-line systemic treatments among stage IV NSCLC patients in California and compares survival among treatment groups. METHODS: Data on 17 254 patients diagnosed with stage IV NSCLC from 2012 to 2014 were obtained from the California Cancer Registry. Systemic treatments were classified into six groups. The Kaplan-Meier method and multivariable Cox proportional hazards models were used to compare survival between treatment groups. RESULTS: Fifty-one percent of patients were known to have received systemic treatment. For patients with nonsquamous histology, pemetrexed regimens were the most common treatment (14.8%) followed by tyrosine kinase inhibitors (11.9%) and platinum doublets (11.5%). Few patients received pemetrexed/bevacizumab combinations (4.5%), bevacizumab combinations (3.6%), or single agents (1.7%). There was statistically significantly better overall survival for those on pemetrexed regimens (hazard ratio [HR] = 0.86, 95% confidence interval [CI] = 0.80 to 0.92), bevacizumab regimens (HR = 0.73, 95% CI = 0.65 to 0.81), pemetrexed/bevacizumab regimens (HR = 0.68, 95% CI = 0.61 to 0.76), or tyrosine kinase inhibitors (HR = 0.62, 95% CI = 0.57 to 0.67) compared with platinum doublets. The odds of receiving most systemic treatments decreased with decreasing socioeconomic status. For patients with squamous histology, platinum doublets were predominant (33.7%) and were not found to have statistically significantly different overall survival from single agents. CONCLUSIONS: These population-level findings indicate low utilization of systemic treatments, survival differences between treatment groups, and evident treatment disparities by socioeconomic status.

A text-mining approach to obtain detailed treatment information from free-text fields in population-based cancer registries: A study of non-small cell lung cancer in California.

BACKGROUND: Population-based cancer registries have treatment information for all patients making them an excellent resource for population-level monitoring. However, specific treatment details, such as drug names, are contained in a free-text format that is difficult to process and summarize. We assessed the accuracy and efficiency of a text-mining algorithm to identify systemic treatments for lung cancer from free-text fields in the California Cancer Registry. METHODS: The algorithm used Perl regular expressions in SAS 9.4 to search for treatments in 24,845 free-text records associated with 17,310 patients in California diagnosed with stage IV non-small cell lung cancer between 2012 and 2014. Our algorithm categorized treatments into six groups that align with National Comprehensive Cancer Network guidelines. We compared results to a manual review (gold standard) of the same records. RESULTS: Percent agreement ranged from 91.1% to 99.4%. Ranges for other measures were 0.71-0.92 (Kappa), 74.3%-97.3% (sensitivity), 92.4%-99.8% (specificity), 60.4%-96.4% (positive predictive value), and 92.9%-99.9% (negative predictive value). The text-mining algorithm used one-sixth of the time required for manual review. CONCLUSION: SAS-based text mining of free-text data can accurately detect systemic treatments administered to patients and save considerable time compared to manual review, maximizing the utility of the extant information in population-based cancer registries for comparative effectiveness research.

Molecular Testing for the Treatment of Advanced Colorectal Cancer: An Overview.

Concurrent with an expansion in the number of agents available for the treatment of advanced CRC, there has been an increase in our understanding of selection biomarkers to optimize the management of patients with this disease. For CRC patients being considered for anti-EGFR therapy, expanded RAS testing is the standard of care to determine the subset of patients who can benefit from cetuximab or panitumumab in conjunction with chemotherapy. A small fraction of patients have HER2 amplification where emerging data suggest treatment with drugs targeting this alteration. Although advanced CRC patients who harbor the BRAF V600E mutation have a poorer prognosis, they are eligible for combinatorial therapy targeting EGFR/BRAF or BRAF/MEK within the MAP kinase signaling pathway. Once primarily thought to be a negative prognostic marker, BRAF V600E mutation is now considered as a positive predictive factor with an opportunity for clinical intervention. A growing body of evidence also supports MSI testing as clinical benefits with immune checkpoint blockade by cancer immunotherapy have been demonstrated in MSI-high patients whose tumors exhibit high mutational burden. It has been established that UGT1A1*28 polymorphism is associated with irinotecan toxicity, but this test is rarely performed as the management strategy has not been identified. No established predictive biomarker for anti-VEGF therapy has yet to be discovered.It is becoming increasingly apparent that our growing understanding of biomarkers is revolutionizing and improving our strategies in the treatment of advanced CRC. Traditional nonselective cytotoxic chemotherapy is gradually being augmented and even in some cases supplanted by selective targeted agents based on our increasing understanding of tumor signaling and mechanism at the molecular level. The prospect of personalized medicine in directing treatment approaches that are optimally beneficial for patients brings tremendous excitement to the growing field of cancer therapeutics. As discussed in this chapter, the concurrent development of molecular biomarkers with new treatment strategies holds great promise of precision medicine in improving outcomes for patients with advanced CRC.

Functional characterization of small CTD phosphatases.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase (RNAP) II undergoes reversible phosphorylation with each round of transcription essential for the regulation of gene expression. A family of small CTD phosphatases (SCPs) was identified based on a homology search to TFIIF-associating CTD phosphatase 1 (FCP1). Unlike FCP1, SCP preferentially catalyze the dephosphorylation of Ser5 within the CTD and is especially active toward RNAP II phosphorylated by TFIIH (1). Recently, SCP1 was demonstrated as a transcriptional regulator that acts to silence neuronal genes (2). This chapter describes the procedures for various assays involved in the discovery and functional characterization of SCPs.

CTD phosphatase: role in RNA polymerase II cycling and the regulation of transcript elongation.

The repetitive C-terminal domain (CTD) of the largest RNA polymerase II subunit plays a critical role in the regulation of gene expression. The activity of the CTD is dependent on its state of phosphorylation. A variety of CTD kinases act on RNA polymerase II at specific steps in the transcription cycle and preferentially phosphorylate distinct positions within the CTD consensus repeat. A single CTD phosphatase has been identified and characterized that in concert with CTD kinases establishes the level of CTD phosphorylation. The involvement of CTD phosphatase in controlling the progression of RNAP II around the transcription cycle, the mobilization of stored RNAP IIO, and the regulation of transcript elongation and RNA processing is discussed.

Hypercholesterolemia impairs exercise capacity in mice.

We previously reported an attenuation of both exercise hyperemia and measures of aerobic capacity in hypercholesterolemic mice. In this study, we expanded upon the previous findings by examining the temporal and quantitative relationship of hypercholesterolemia to aerobic and anaerobic capacity and by exploring several potential mechanisms of dysfunction. Eight-week-old wild type (n = 123) and apoE knockout (n = 79) C57BL/6J mice were divided into groups with distinct cholesterol levels by feeding with regular or high-fat diets. At various ages, the mice underwent treadmill ergospirometry. To explore mechanisms, aortic ring vasodilator function and nitrate (NO(x)) activity, urinary excretion of NO(x), running muscle microvascular density and citrate synthase activity, as well as myocardial mass and histologic evidence of ischemia were measured. At 8 weeks of age, all mice had similar measures of exercise capacity. All indices of aerobic exercise capacity progressively declined at 12 and 20 weeks of age in the hypercholesterolemic mice as cholesterol levels increased while indices of anaerobic capacity remained unaffected. Across the four cholesterol groups, the degree of aerobic dysfunction was related to serum cholesterol levels; a relationship that was maintained after correcting for confounding factors. Associated with the deterioration in exercise capacity was a decline in measures of nitric oxide-mediated vascular function while there was no evidence of aberrations in functional or oxidative capacities or in other components of transport capacity. In conclusion, aerobic exercise dysfunction is observed in murine models of genetic and diet-induced hypercholesterolemia and is associated with a reduction in vascular nitric oxide production.

A kinase-independent function of c-Abl in promoting proteolytic destruction of damaged DNA binding proteins.

Damaged DNA binding proteins (DDBs) play a critical role in the initial recognition of UV-damaged DNA and mediate recruitment of nucleotide excision repair factors. Previous studies identified DDB2 as a target of the CUL-4A ubiquitin ligase. However, the biochemical mechanism governing DDB proteolysis and its underlying physiological function in the removal of UV-induced DNA damage are largely unknown. Here, we report that the c-Abl nonreceptor tyrosine kinase negatively regulates the repair of UV-induced photolesions on genomic DNA. Biochemical studies revealed that c-Abl promotes CUL-4A-mediated DDB ubiquitination and degradation in a manner that does not require its tyrosine kinase activity both under normal growth conditions and following UV irradiation. Moreover, c-Abl activates DDB degradation in part by alleviating the inhibitory effect of CAND1/TIP120A on CUL-4A. These results revealed a kinase-independent function of c-Abl in a ubiquitin-proteolytic pathway that regulates the damage recognition step of nucleotide excision repair.

TFIIF-associating carboxyl-terminal domain phosphatase dephosphorylates phosphoserines 2 and 5 of RNA polymerase II.

The carboxyl-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit undergoes reversible phosphorylation throughout the transcription cycle. The unphosphorylated form of RNAP II is referred to as IIA, whereas the hyperphosphorylated form is known as IIO. Phosphorylation occurs predominantly at serine 2 and serine 5 within the CTD heptapeptide repeat and has functional implications for RNAP II with respect to initiation, elongation, and transcription-coupled RNA processing. In an effort to determine the role of the major CTD phosphatase (FCP1) in regulating events in transcription that appear to be influenced by serine 2 and serine 5 phosphorylation, the specificity of FCP1 was examined. FCP1 is capable of dephosphorylating heterogeneous RNAP IIO populations of HeLa nuclear extracts. The extent of dephosphorylation at specific positions was assessed by immunoreactivity with monoclonal antibodies specific for phosphoserine 2 or phosphoserine 5. As an alternative method to assess FCP1 specificity, RNAP IIO isozymes were prepared in vitro by the phosphorylation of purified calf thymus RNAP IIA with specific CTD kinases and used as substrates for FCP1. FCP1 dephosphorylates serine 2 and serine 5 with comparable efficiency. Accordingly, the specificity of FCP1 is sufficiently broad to dephosphorylate RNAP IIO at any point in the transcription cycle irrespective of the site of serine phosphorylation within the consensus repeat.

The repetitive C-terminal domain of RNA polymerase II: multiple conformational states drive the transcription cycle.

RNA polymerase (RNAP) II is a complex multisubunit enzyme responsible for the synthesis of mRNA in eukaryotic cells. The largest subunit contains at its C-terminus a unique domain, designated the CTD, comprised of tandem repeats of the consensus sequence Tyr(1)Ser(2)Pro(3)Thr(4)Ser(5)Pro(6)Ser(7). This repeat occurs 52 times in mammalian RNAP II. The CTD is subject to extensive phosphorylation at specific points in the transcription cycle by distinct CTD kinases that phosphorylate certain positions within the consensus repeat. The level and pattern of phosphorylation is determined by the concerted action of CTD kinases and CTD phosphatases. The highly dynamic modification by multiple CTD kinases and phosphatases generate distinct conformations of the CTD that facilitate the recruitment of specific macromolecular assemblies to RNAP II. These CTD interacting proteins influence formation of a preinitiation complex at the promoter and couple processing of the primary transcript to the elongation complex.

A novel RNA polymerase II C-terminal domain phosphatase that preferentially dephosphorylates serine 5.

The transcription and processing of pre-mRNA in eukaryotic cells are regulated in part by reversible phosphorylation of the C-terminal domain of the largest RNA polymerase (RNAP) II subunit. The CTD phosphatase, FCP1, catalyzes the dephosphorylation of RNAP II and is thought to play a major role in polymerase recycling. This study describes a family of small CTD phosphatases (SCPs) that preferentially catalyze the dephosphorylation of Ser5 within the consensus repeat. The preferred substrate for SCP1 is RNAP II phosphorylated by TFIIH. Like FCP1, the activity of SCP1 is enhanced by the RAP74 subunit of TFIIF. Expression of SCP1 inhibits activated transcription from a number of promoters, whereas a phosphatase-inactive mutant of SCP1 enhances transcription. Accordingly, SCP1 may play a role in the regulation of gene expression, possibly by controlling the transition from initiation/capping to processive transcript elongation.

NOS inhibition accelerates atherogenesis: reversal by exercise.

In this study, we assessed the effects of chronic exercise training (12 wk) on atherosclerotic lesion formation in hypercholesterolemic apolipoprotein E-deficient mice (n = 31). At the age of 9 wk, mice were assigned to the following groups: sedentary (Sed; n = 9); exercise (Ex; n = 12); sedentary and oral NG-nitro-L-arginine (L-NNA, Sed-NA; n = 4), or exercise and oral L-NNA (Ex-NA; n = 6). Chronic exercise training was performed on a treadmill for 12 wk (6 times/wk and twice for 1 h/day) at a final speed of 22 m/min, and an 8 degrees grade. L-NNA was discontinued 5 days before final treadmill testing. The farthest distance run to exhaustion was observed in Ex-NA mice (Sed: 306 +/- 32 m; Ex: 640 +/- 87; Sed-NA: 451 +/- 109 m; Ex-NA: 820 +/- 49 m; all P < 0.05). Lesion formation was assessed in the proximal ascending aorta by dissection microscopy after oil red O staining. The aortas of Sed-NA mice manifested a threefold increase in lesion formation compared with the other groups. This L-NNA-induced lesion formation was reduced by chronic exercise training (Sed, 786 +/- 144; Ex, 780 +/- 206; Sed-NA, 2,147 +/- 522; Ex-NA, 851 +/- 253; Sed-NA vs. all other groups: P < 0.001). In conclusion, treatment with oral L-NNA (an nitric oxide synthase antagonist) leads to accelerated atherogenesis in genetically determined hypercholesterolemic mice. This adverse effect can be overcome by chronic exercise training.