Associations between Plasma Tocopherols and Lung Cancer Risk: Results from the Southern Community Cohort Study.

BACKGROUND: Despite the various anticancer activities of tocopherols, little is known about tocopherols associated with lung cancer risk among low-income African Americans (AA) and European Americans (EA) who are disproportionately affected by the disease. METHODS: We conducted a nested case-control study that included 209 incident lung cancer cases and 406 matched controls within the Southern Community Cohort Study. Using biospecimens collected at cohort enrollment, plasma levels of α-, ß/γ-, δ-, and total-tocopherols were measured by high-performance liquid chromatography with photodiode array detection. Conditional logistic regression was used to estimate ORs and 95% confidence intervals (CI) for lung cancer risk after adjusting for potential confounders. Stratified analyses were also conducted. RESULTS: Plasma levels of total-tocopherols were inversely associated with lung cancer risk overall [OR (95% CI) for the highest vs. lowest tertile = 0.51 (0.30-0.90)]. The inverse association remained significant among EAs [0.20 (0.06-0.65)], men [0.43 (0.21-0.90)], current smokers [0.49 (0.26-0.93)], and cases diagnosed within 2 years of blood draw [0.36 (0.15-0.86)], though we did not find a significant risk reduction among AAs [0.75 (0.39-1.45)]. Notably, we found significant interactions between α-tocopherol and race after controlling the FDR to correct for multiple comparisons (Pinteraction = 0.02). CONCLUSIONS: Our results indicate that plasma total-tocopherols are inversely associated with lung cancer risk, but the association may differ across specific isomeric forms of tocopherols, race, or other individuals' characteristics. Further large-scale studies are warranted to confirm our findings. IMPACT: Recommendations on tocopherols for lung cancer prevention should take isomers, race, and smoking behaviors into consideration.

Pre-diagnostic circulating p53 autoantibodies and subsequent lung cancer risk in low-income African and European Americans.

INTRODUCTION: Mutations of the TP53 gene lead to the production of autoantibodies against p53, a major tumor suppressor protein. Although studies have indicated the association of p53 autoantibodies with human cancers, epidemiologic evidence on lung cancer is still lacking. METHODS: In this nested case-control study conducted within the Southern Community Cohort Study, we investigated the association of circulating p53 autoantibodies with the subsequent risk of developing lung cancer. Using blood samples collected prior to any cancer diagnosis from 295 cases and their individually matched controls, seroreactivity to p53 was assessed by fluorescent bead-based multiplex serology. Conditional logistic regression models were used to estimate odds ratios (OR) and 95 % confidence intervals (CI) for lung cancer risk associated with p53 autoantibodies. RESULTS: After adjustment for potential confounders, p53 seropositivity was significantly associated with an increased risk of lung cancer (OR=2.98, 95 % CI: 1.10-8.06) among African Americans, but not among European Americans (OR=1.21, 95 % CI: 0.24-6.15). The positive associations were restricted to men (OR=4.59, 95 % CI: 1.30-16.16) and participants with a short interval (≤ 4 years) from blood collection to diagnosis (OR=4.30, 95 % CI: 1.33-13.89). CONCLUSION: Our findings add to the evidence supporting p53 autoantibodies as a biomarker of lung cancer.

Associations of Pre-Diagnostic Serum Levels of Total Bilirubin and Albumin With Lung Cancer Risk: Results From the Southern Community Cohort Study.

Background: Previous studies conducted among European and Asian decedents reported inverse associations of serum total bilirubin and albumin with lung cancer risk. Yet, no study has been conducted among African Americans or low-income European Americans. Methods: This study included 522 incident lung cancer cases and 979 matched controls nested in the Southern Community Cohort Study, a cohort of predominantly low-income African and European Americans. Serum levels of total bilirubin and albumin, collected up to 11 years prior to case diagnoses, were measured by a clinical chemistry analyzer. Conditional logistic regression models were applied to evaluate the associations of total bilirubin and albumin with lung cancer risk. Results: Overall, serum levels of total bilirubin (ORT3 vs. T1 = 0.96, 95% CI: 0.66-1.39) were not significantly associated with lung cancer risk. However, higher levels of serum total bilirubin were significantly associated with decreased risk of lung cancer among participants who were diagnosed within two years following sample collection (ORT3 vs. T1 = 0.36, 95% CI: 0.15-0.87) and among former/never smokers (ORT3 vs. T1 = 0.54, 95% CI: 0.32-0.93). Serum levels of albumin were significantly associated with decreased risk of lung cancer overall (ORT3 vs. T1 = 0.70, 95% CI: 0.50-0.98) and among African Americans (ORT3 vs. T1 = 0.62, 95% CI: 0.41-0.96), but not among European Americans. Conclusion: Our results indicate that in a low-income African American and European American population, serum levels of total bilirubin may be related to lung cancer progression and differ by smoking status. Meanwhile, the association of serum albumin levels with lung cancer risk may differ by race. Further studies are warranted to confirm these results.

Associations of lung cancer risk with biomarkers of Helicobacter pylori infection.

Helicobacter pylori infection has been suggested to be associated with lung cancer risk. However, information is lacking on whether the association differs by H. pylori antigen. We conducted a nested case-control study within the Southern Community Cohort Study, including 295 incident lung cancer cases and 295 controls. Helicobacter pylori multiplex serology assay was performed to detect antibodies to 15 H. pylori proteins. Conditional logistic regression was used to estimate odds ratios (ORs) and confidence intervals (95% CIs) after adjustment for covariates. Overall H. pylori+ was associated with a non-statistically significant increased risk of lung cancer (OR: 1.29; 95% CI: 0.85-1.95). Significant associations, however, were observed for H. pylori+ VacA+ (OR: 1.64; 95% CI: 1.02-2.62) and H. pylori+ Catalase+ (OR: 1.75; 95% CI: 1.11-2.77). The positive association of H. pylori+ Catalase+ with lung cancer risk was predominantly seen among African Americans (OR: 2.09; 95% CI: 1.11-3.95) but not European Americans (OR: 1.20; 95% CI: 0.56-2.54). Among participants who smoked ≥ 30 pack-years, overall H. pylori+ (OR: 1.85; 95% CI: 1.02-3.35), H. pylori+ CagA+ (OR: 2.77; 95% CI: 1.35-5.70), H. pylori+ VacA+ (OR: 2.53; 95% CI: 1.25-5.13) and H. pylori+ HP1564+ (OR: 2.01; 95% CI: 1.07-3.77) were associated with increased risk of lung cancer. Our study provides novel evidence that associations of H. pylori infection with lung cancer risk differ by H. pylori biomarker, may be more evident among African Americans and may be modified by smoking habits. Furthermore, studies are warranted to confirm our findings.

The Role of Thoracic Radiation Therapy Dosing in the Treatment of Limited-Stage Small Cell Lung Cancer: A Study Based on the National Cancer Database.

Purpose: Small cell lung cancer (SCLC) is a highly fatal disease, but its treatment has remained relatively unchanged for decades. Randomized clinical trials evaluating radiation therapy (RT) dosing and fractionation have yielded mixed results on overall survival (OS). Methods and Materials: We identified 2261 patients with limited-stage (LS) SCLC undergoing definitive RT at 1.5, 1.8, and 2.0 Gy dose per fraction, concurrently with chemotherapy, between 2004 and 2015 within the National Cancer Database. Overall survival (OS) was evaluated using the Kaplan-Meier method, and Cox proportional hazards regression was used to investigate whether there was any survival difference among patients who received hyperfractionated, twice-daily RT at 1.5 Gy per fraction (HF1.5) and once-daily, standard fractionation RT at 1.8 Gy (SF1.8) or 2.0 Gy (SF2.0) per fraction. Subgroup analyses by age, sex, race, time to RT, facility type, and Charlson comorbidity index were also performed. Results: All stage median OS rates for HF1.5, SF1.8, and SF2.0 Gy groups were 21.6, 18.9, and 19.4 months, respectively (log-rank P = .0079). Multivariate analyses adjusting for demographic factors, socioeconomic status, tumor characteristics, and year of diagnosis showed SF1.8 (hazard ratio [HR] = 1.30, 1.03-1.63) and SF2.0 (HR = 1.20, 1.00-1.45) was associated with worse 1-year survival compared with HF1.5. This association was more evident in stage IIb-stage III than stage I to stage IIa patients. Propensity score-weighted analysis showed similar results. Stratified analyses showed the significant associations were confined to male or black patients, those aged >65 years, with 1 comorbidity, who had waited >60 days to start RT or were treated at an academic medical center. Conclusions: Analyses of real-world treatment outcome data showed that receiving hyperfractionated, twice-daily RT was associated with improved survival among patients with LS-SCLC compared with standard, once-daily fractionation regimens at 1 year after diagnosis, particularly for subsets of patients. Some associations retained statistical significance 3 years postdiagnosis.

TBX1 functions as a putative oncogene of breast cancer through promoting cell cycle progression.

We have previously identified a genetic variant, rs34331122 in the 22q11.21 locus, as being associated with breast cancer risk in a genome-wide association study. This novel variant is located in the intronic region of the T-box transcription factor 1 (TBX1) gene. Cis-expression quantitative trait loci analysis showed that expression of TBX1 was regulated by the rs34331122 variant. In the current study, we investigated biological functions and potential molecular mechanisms of TBX1 in breast cancer. We found that TBX1 expression was significantly higher in breast cancer tumor tissues than adjacent normal breast tissues and increased with tumor stage (P < 0.05). We further knocked-down TBX1 gene expression in three breast cancer cell lines, MDA-MB-231, MCF-7 and T47D, using small interfering RNAs and examined consequential changes on cell oncogenicity and gene expression. TBX1 knock-down significantly inhibited breast cancer cell proliferation, colony formation, migration and invasion. RNA sequencing and flow cytometry analysis revealed that TBX1 knock-down in breast cancer cells induced cell cycle arrest in the G1 phase through disrupting expression of genes involved in the cell cycle pathway. Furthermore, survival analysis using the online Kaplan-Meier Plotter suggested that higher TBX1 expression was associated with worse outcomes in breast cancer patients, especially for estrogen receptor-positive breast cancer, with HRs (95% CIs) for overall survival (OS) and distant metastasis free survival (DMFS) of 1.5 (1.05-2.15) and 1.55 (1.10-2.18), respectively. In conclusion, our results suggest that the TBX1 gene may act as a putative oncogene of breast cancer through regulating expressions of cell cycle-related genes.

Functional Genomic Analyses of the 21q22.3 Locus Identifying Functional Variants and Candidate Gene YBEY for Breast Cancer Risk.

We previously identified a locus at 21q22.3, tagged by the single nucleotide polymorphism (SNP) rs35418111, being associated with breast cancer risk at a genome-wide significance level; however, the underlying causal functional variants and gene(s) responsible for this association are unknown. We performed functional genomic analyses to identify potential functional variants and target genes that may mediate this association. Functional annotation for SNPs in high linkage disequilibrium (LD, r2 > 0.8) with rs35418111 in Asians showed evidence of promoter and/or enhancer activities, including rs35418111, rs2078203, rs8134832, rs57385578, and rs8126917. These five variants were assessed for interactions with nuclear proteins by electrophoretic mobility shift assays. Our results showed that the risk alleles for rs2078203 and rs35418111 altered DNA-protein interaction patterns. Cis-expression quantitative loci (cis-eQTL) analysis, using data from the Genotype-Tissue Expression database (GTEx) European-ancestry female normal breast tissue, indicated that the risk allele of rs35418111 was associated with a decreased expression of the YBEY gene, a relatively uncharacterized endoribonuclease in humans. We investigated the biological effects of YBEY on breast cancer cell lines by transient knock-down of YBEY expression in MCF-7, T47D, and MDA-MB-231 cell lines. Knockdown of YBEY mRNA in breast cancer cell lines consistently decreased cell proliferation, colony formation, and migration/invasion, regardless of estrogen receptor status. We performed RNA sequencing in MDA-MB-231 cells transfected with siRNA targeting YBEY and subsequent gene set enrichment analysis to identify gene networks associated with YBEY knockdown. These data indicated YBEY was involved in networks associated with inflammation and metabolism. Finally, we showed trends in YBEY expression patterns in breast tissues from The Cancer Genome Atlas (TCGA); early-stage breast cancers had elevated YBEY expression compared with normal tissue, but significantly decreased expression in late-stage disease. Our study provides evidence of a significant role for the human YBEY gene in breast cancer pathogenesis and the association between the rs35418111/21q22.3 locus and breast cancer risk, which may be mediated through functional SNPs, rs35418111 and rs2078203, that regulate expression of YBEY.

Prospective study of plasma levels of coenzyme Q10 and lung cancer risk in a low-income population in the Southeastern United States.

BACKGROUND: Coenzyme Q10 (CoQ10) is a ubiquitous molecule in living organisms serving as a cofactor in energy production. Epidemiological studies have reported low CoQ10 levels being associated with an increased risk of various cancers. We conducted the first study to evaluate the association of CoQ10 concentrations with lung cancer risk. METHODS: A nested case-control study including 201 lung cancer cases and 395 matched controls from the Southern Community Cohort Study was conducted. Plasma CoQ10 levels were measured using high-performance liquid chromatography with photo-diode array detection. Conditional logistic regression models were applied to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between plasma CoQ10 levels and lung cancer risk. RESULTS: Plasma CoQ10 concentration was inversely associated with the risk of lung cancer. After adjusting for age, sex, race, and socioeconomic status, the OR (95% CI) comparing the third to first tertile was 0.57 (0.36-0.91, P for trend = 0.02). Further adjustments for smoking, alcohol, chronic obstructive pulmonary disease, and body mass index attenuated the point estimate slightly (OR = 0.60, 95% CI = 0.34-1.08, P for trend = 0.11), comparing third to first tertiles. Stratified analyses identified a significant inverse association between plasma CoQ10 levels and lung cancer risk in current smokers, but not in former/never smokers. The association was more evident in cases who were diagnosed within 1 year of blood draw than in cases diagnosed after 1 year. CONCLUSIONS: Low plasma CoQ10 was significantly associated with increased lung cancer risk, particularly among current smokers. The stronger association seen shortly following the blood draw suggests that CoQ10 may be related to disease progression.

MicroRNA-92 Expression in CD133+ Melanoma Stem Cells Regulates Immunosuppression in the Tumor Microenvironment via Integrin-Dependent Activation of TGFß.

In addition to being refractory to treatment, melanoma cancer stem cells (CSC) are known to suppress host antitumor immunity, the underlying mechanisms of which need further elucidation. In this study, we established a novel role for miR-92 and its associated gene networks in immunosuppression. CSCs were isolated from the B16-F10 murine melanoma cell line based on expression of the putative CSC marker CD133 (Prominin-1). CD133+ cells were functionally distinct from CD133- cells and showed increased proliferation in vitro and enhanced tumorigenesis in vivo. CD133+ CSCs also exhibited a greater capacity to recruit immunosuppressive cell types during tumor formation, including FoxP3+ Tregs, myeloid-derived suppressor cells (MDSC), and M2 macrophages. Using microarray technology, we identified several miRs that were significantly downregulated in CD133+ cells compared with CD133- cells, including miR-92. Decreased expression of miR-92 in CSCs led to higher expression of target molecules integrin αV and α5 subunits, which, in turn, enhanced TGFß activation, as evidenced by increased phosphorylation of SMAD2. CD133+ cells transfected with miR-92a mimic and injected in vivo showed significantly decreased tumor burden, which was associated with reduced immunosuppressive phenotype intratumorally. Using The Cancer Genome Atlas database of patients with melanoma, we also noted a positive correlation between integrin α5 and TGFß1 expression levels and an inverse association between miR-92 expression and integrin alpha subunit expression. Collectively, this study suggests that a miR-92-driven signaling axis involving integrin activation of TGFß in CSCs promotes enhanced tumorigenesis through induction of intratumoral immunosuppression. SIGNIFICANCE: CD133+ cells play an active role in suppressing melanoma antitumor immunity by modulating miR-92, which increases influx of immunosuppressive cells and TGFß1 expression.

Development of the plant-derived peptide lunasin as an anticancer agent.

The health benefits of soy consumption have long been recognized. An important potential benefit is the linkage of soy consumption with reduced cancer risk. One emerging factor that may confer the anticancer effects of soy is the peptide lunasin. Lunasin has both chemopreventive and therapeutic activities against a variety of carcinogens and cancer types. A novel feature of lunasin is that it contains multiple functional domains that can modulate gene expression through effects on histone acetylation and integrin signaling. Recent studies suggest that lunasin effects on integrin signaling in cancer stem cells reduce expression of stemness factors with a concomitant reduction in metastatic potential. Here, we highlight recent studies of the potential use of lunasin as an anticancer agent and its mode of action.

The soy-derived peptide Lunasin inhibits invasive potential of melanoma initiating cells.

Lunasin is a 44 amino acid peptide with multiple functional domains including an aspartic acid tail, an RGD domain, and a chromatin-binding helical domain. We recently showed that Lunasin induced a phenotype switch of cancer initiating cells (CIC) out of the stem compartment by inducing melanocyte-associated differentiation markers while simultaneously reducing stem-cell-associated transcription factors. In the present study, we advance the hypothesis that Lunasin can reduce pools of melanoma cells with stem cell-like properties, and demonstrate that Lunasin treatment effectively inhibits the invasive potential of CICs in vitro as well as in vivo in a mouse experimental metastasis model. Mice receiving Lunasin treatment had significantly reduced pulmonary colonization after injection of highly metastatic B16-F10 melanoma cells compared to mice in the control group. Mechanistic studies demonstrate that Lunasin reduced activating phosphorylations of the intracellular kinases FAK and AKT as well as reduced histone acetylation of lysine residues in H3 and H4 histones. Using peptides with mutated activity domains, we functionally demonstrated that the RGD domain is necessary for Lunasin uptake and its ability to inhibit oncosphere formation by CICs, thus confirming that Lunasin's ability to affect CICs is at least in part due to the suppression of integrin signaling. Our studies suggest that Lunasin represents a unique anticancer agent that could be developed to help prevent metastasis and patient relapse by reducing the activity of CICs which are known to be resistant to current chemotherapies.

Lunasin is a novel therapeutic agent for targeting melanoma cancer stem cells.

Recent studies provide compelling evidence that melanoma is initiated and maintained by a small population of malignant cells called cancer-initiating cells (CICs) that exhibit stem-cell-like properties. Observations that CICs have a distinct biology when compared to that of the bulk tumor cells and, importantly, are resistant to chemotherapies and radiation, suggest that CICs are involved in invasion, metastasis, and ultimately relapse. Lunasin, a bioactive peptide present in soybean, has both chemopreventive activity and chemotherapeutic activity against multiple cancer types. In this study, we tested the potential of Lunasin to specifically target CICs in melanoma tumor cell populations. In vitro studies using human melanoma cell lines showed that Lunasin treatment decreased the size of a subpopulation of melanoma cells expressing the surrogate CIC marker, Aldehyde Dehydrogenase, concomitant with a reduction in the ability to form colonies in soft agar assays, and reduced tumor growth in mouse xenografts. Similarly, Lunasin inhibited colony formation by isolated melanoma CICs in soft agar and reduced oncosphere formation in vitro and substantially inhibited tumor growth in mouse xenografts. Mechanistic studies revealed that Lunasin treatment of isolated melanoma CICs induced expression of the melanocyte-associated differentiation markers Tyrosinase and Microphthalmia-associated Transcription Factor concomitant with reduced expression of the stemness factor NANOG. These findings document for the first time that Lunasin has significant therapeutic activity against melanoma by specifically targeting melanoma CICs, and inducing a more differentiated, non-CIC phenotype. Thus, Lunasin may represent a novel therapeutic option for both chemoresistant and advanced metastatic melanoma management.

Validation of syngeneic mouse models of melanoma and non-small cell lung cancer for investigating the anticancer effects of the soy-derived peptide Lunasin.

Background : Lunasin is a naturally occurring peptide present in soybean that has both chemopreventive and therapeutic activities that can prevent cellular transformation and inhibit the growth of several human cancer types. Recent studies indicate that Lunasin has several distinct potential modes of action including suppressing integrin signaling and epigenetic effects driven by modulation of histone acetylation. In addition to direct effects on cancer cells, Lunasin also has effects on innate immunity that may contribute to its ability to inhibit tumor growth in vivo. Methods: Standard assays for cell proliferation and colony formation were used to assess Lunasin's in vitro activity against murine Lewis lung carcinoma (LLC) and B16-F0 melanoma cells.  Lunasin's in vivo activity was assessed by comparing the growth of tumors initiated by subcutaneous implantation of LLC or B16-F0 cells in Lunasin-treated and untreated C57BL/6 mice. Results: Lunasin was found to inhibit growth of murine LLC cells and murine B16-F0 melanoma cells in vitro and in wild-type C57BL/6 mice.  The effects of Lunasin in these two mouse models were very similar to those previously observed in studies of human non-small cell lung cancer and melanoma cell lines. Conclusions: We have now validated two established syngeneic mouse models as being responsive to Lunasin treatment.  The validation of these two in vivo syngeneic models will allow detailed studies on the combined therapeutic and immune effects of Lunasin in a fully immunocompetent mouse model.