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

Validation of syngeneic mouse models of melanoma and non-small cell lung cancer for investigating the anticancer effects of the soy-derived peptide Lunasin [version 2; referees: 1 approved, 2 approved with reservations]

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

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

Supplementary Table 4. from Associations between Plasma Tocopherols and Lung Cancer Risk: Results from the Southern Community Cohort Study

Association between Plasma Tocopherols and Lung Cancer Risk by Smoking Pack-Years</p

Supplementary Table 5. from Associations between Plasma Tocopherols and Lung Cancer Risk: Results from the Southern Community Cohort Study

Association between Plasma Tocopherols and Lung Cancer Risk by Use of Dietary Supplements</p

Supplementary Figure 1. from Associations between Plasma Tocopherols and Lung Cancer Risk: Results from the Southern Community Cohort Study

Correlation Matrix between Plasma Tocopherols Description. Pearson correlation coefficients (r) were calculated after log-transformation of plasma levels of each tocopherol subtype.</p

Supplementary Table 6. from Associations between Plasma Tocopherols and Lung Cancer Risk: Results from the Southern Community Cohort Study

Association between Plasma Tocopherols and Histological Subtype of Lung Cancer</p

Supplementary Table 2. from Associations between Plasma Tocopherols and Lung Cancer Risk: Results from the Southern Community Cohort Study

Association between Plasma Tocopherols and Lung Cancer Risk by Race</p

Supplementary Table 7. from Associations between Plasma Tocopherols and Lung Cancer Risk: Results from the Southern Community Cohort Study

Association between Plasma Tocopherols and Lung Cancer Risk in Sequential Adjustment Models</p