Heavy-metal associated breast cancer and colorectal cancer hot spots and their demographic and socioeconomic characteristics.

PURPOSE: Cancer registries offer an avenue to identify cancer clusters across large populations and efficiently examine potential environmental harms affecting cancer. The role of known metal carcinogens (i.e., cadmium, arsenic, nickel, chromium(VI)) in breast and colorectal carcinogenesis is largely unknown. Historically marginalized communities are disproportionately exposed to metals, which could explain cancer disparities. We examined area-based metal exposures and odds of residing in breast and colorectal cancer hotspots utilizing state tumor registry data and described the characteristics of those living in heavy metal-associated cancer hotspots. METHODS: Breast and colorectal cancer hotspots were mapped across Kentucky, and area-based ambient metal exposure to cadmium, arsenic, nickel, and chromium(VI) were extracted from the 2014 National Air Toxics Assessment for Kentucky census tracts. Among colorectal cancer (n = 56,598) and female breast cancer (n = 77,637) diagnoses in Kentucky, we used logistic regression models to estimate Odds Ratios (ORs) and 95% Confidence Intervals to examine the association between ambient metal concentrations and odds of residing in cancer hotspots, independent of individual-level and neighborhood risk factors. RESULTS: Higher ambient metal exposures were associated with higher odds of residing in breast and colorectal cancer hotspots. Populations in breast and colorectal cancer hotspots were disproportionately Black and had markers of lower socioeconomic status. Furthermore, adjusting for age, race, tobacco and neighborhood factors did not significantly change cancer hotspot ORs for ambient metal exposures analyzed. CONCLUSION: Ambient metal exposures contribute to higher cancer rates in certain geographic areas that are largely composed of marginalized populations. Individual-level assessments of metal exposures and cancer disparities are needed.

miR-186 induces tetraploidy in arsenic exposed human keratinocytes.

Chronic inorganic arsenic (iAs) exposure in drinking water is a global issue affecting >225 million people. Skin is a major target organ for iAs. miRNA dysregulation and chromosomal instability (CIN) are proposed mechanisms of iAs-induced carcinogenesis. CIN is a cancer hallmark and tetraploid cells can better tolerate increase in chromosome number and aberration, contributing to the evolution of CIN. miR-186 is overexpressed in iAs-induced squamous cell carcinoma relative to iAs-induced hyperkeratosis. Bioinformatic analysis indicated that miR-186 targets mRNAs of important cell cycle regulators including mitotic checkpoint serine/threonine kinase B (BUB1) and cell division cycle 27 (CDC27). We hypothesized that miR-186 overexpression contributes to iAs-induced transformation of keratinocytes by targeting mitotic regulators leading to induction of CIN. Ker-CT cells, a near diploid human keratinocyte cell line, were transduced with miR-186 overexpressing or scrambled control lentivirus. Stable clones were isolated after puromycin selection. Clones transduced with lentivirus expressing either a scrambled control miRNA or miR-186 were maintained with 0 or 100 nM iAs for 4 weeks. Unexposed scrambled control clones were considered as passage matched controls. Chronic iAs exposure increased miR-186 expression in miR-186 clones. miR-186 overexpression significantly reduced CDC27 levels irrespective of iAs exposure. The percentage of tetraploid or aneuploid cells was increased in iAs exposed miR-186 clones. Aneuploidy can arise from a tetraploid intermediate. Suppression of CDC27 by miR-186 may lead to impairment of mitotic checkpoint complex formation and its ability to maintain cell cycle arrest leading to chromosome misalignment. As a result, cells overexpressing miR-186 and chronically exposed to iAs may have incorrect chromosome segregation and CIN. These data suggest that dysregulation of miRNA by iAs mediates tetraploidy, aneuploidy and chromosomal instability contributing to iAs-induced carcinogenesis.

miRNA dysregulation is an emerging modulator of genomic instability.

Genomic instability consists of a range of genetic alterations within the genome that contributes to tumor heterogeneity and drug resistance. It is a well-established characteristic of most cancer cells. Genome instability induction results from defects in DNA damage surveillance mechanisms, mitotic checkpoints and DNA repair machinery. Accumulation of genetic alterations ultimately sets cells towards malignant transformation. Recent studies suggest that miRNAs are key players in mediating genome instability. miRNAs are a class of small RNAs expressed in most somatic tissues and are part of the epigenome. Importantly, in many cancers, miRNA expression is dysregulated. Consequently, this review examines the role of miRNA dysregulation as a causal step for induction of genome instability and subsequent carcinogenesis. We focus specifically on mechanistic studies assessing miRNA(s) and specific subtypes of genome instability or known modes of genome instability. In addition, we provide insight on the existing knowledge gaps within the field and possible ways to address them.

Chronic arsenic exposure suppresses ATM pathway activation in human keratinocytes.

An estimated 220 million people worldwide are chronically exposed to inorganic arsenic (iAs) primarily as a result of drinking iAs-contaminated water. Chronic iAs exposure is associated with a plethora of human diseases including skin lesions and multi-organ cancers. iAs is a known clastogen, inducing DNA double strand breaks (DSBs) in both exposed human populations and in vitro. However, iAs does not directly interact with DNA, suggesting that other mechanisms, such as inhibition of DNA repair and DNA Damage Response (DDR) signaling, may be responsible for iAs-induced clastogenesis. Recent RNA-sequencing data from human keratinocytes (HaCaT cells) indicate that mRNAs for phosphatases important for resolution of DDR signaling are induced as a result of chronic iAs exposure prior to epithelial to mesenchymal transition. Here, we report that phosphorylation of ataxia telengectasia mutated (ATM) protein at a critical site (pSer1981) important for DDR signaling, and downstream CHEK2 activation, are significantly reduced in two human keratinocyte lines as a result of chronic iAs exposure. Moreover, RAD50 expression is reduced in both of these lines, suggesting that suppression of the MRE11-RAD50-NBS1 (MRN) complex may be responsible for reduced ATM activation. Lastly, we demonstrate that DNA double strand break accumulation and DNA damage is significantly higher in human keratinocytes with low dose iAs exposure. Thus, inhibition of the MRN complex in iAs-exposed cells may be responsible for reduced ATM activation and reduced DSB repair by homologous recombination (HR). As a result, cells may favor error-prone DSB repair pathways to fix damaged DNA, predisposing them to chromosomal instability (CIN) and eventual carcinogenesis often seen resulting from chronic iAs exposure.

Chronic arsenic exposure suppresses proteasomal and autophagic protein degradation.

Ubiquitin Proteasomal System (UPS) and autophagy dysregulation initiate cancer. These pathways are regulated by zinc finger proteins. Trivalent inorganic arsenic (iAs) displaces zinc from zinc finger proteins disrupting functions of important cellular proteins. The effect of chronic environmental iAs exposure (100 nM) on UPS has not been studied. We tested the hypothesis that environmental iAs exposure suppresses UPS, activating autophagy as a compensatory mechanism. We exposed skin (HaCaT and Ker-CT; independent quadruplicates) and lung (BEAS-2B; independent triplicates) cell cultures to 0 or 100 nM iAs for 7 or 8 weeks. We quantified ER stress (XBP1 splicing employing Reverse Transcriptase -Polymerase Chain Reaction), proteasomal degradation (immunoblots), and initiation and completion of autophagy (immunoblots). We demonstrate that chronic iAs exposure suppresses UPS, initiates autophagy, but suppresses autophagic protein degradation in skin and lung cell lines. Our data suggest that chronic iAs exposure inhibits autophagy which subsequently suppresses UPS.

miRNAs and arsenic-induced carcinogenesis.

Arsenic-induced carcinogenesis is a worldwide health problem. Identifying the molecular mechanisms responsible for the induction of arsenic-induced cancers is important for developing treatment strategies. MicroRNA (miRNA) dysregulation is known to affect development and progression of human cancer. Several studies have identified an association between altered miRNA expression in cancers from individuals chronically exposed to arsenic and in cell models for arsenic-induced carcinogenesis. This chapter provides a comprehensive review for miRNA dysregulation in arsenic-induced cancer.

Human macrophage-engineered vesicles for utilization in ovarian cancer treatment.

Background: Ovarian cancer is a deadly female malignancy with a high rate of recurrent and chemotherapy-resistant disease. Tumor-associated macrophages (TAMs) are a significant component of the tumor microenvironment and include high levels of M2-protumor macrophages that promote chemoresistance and metastatic spread. M2 macrophages can be converted to M1 anti-tumor macrophages, representing a novel therapeutic approach. Vesicles engineered from M1 macrophages (MEVs) are a novel method for converting M2 macrophages to M1 phenotype-like macrophages. Methods: Macrophages were isolated and cultured from human peripheral blood mononuclear cells. Macrophages were stimulated to M1 or M2 phenotypes utilizing LPS/IFN-γ and IL-4/IL-13, respectively. M1 MEVs were generated with nitrogen cavitation and ultracentrifugation. Co-culture of ovarian cancer cells with macrophages and M1 MEVs was followed by cytokine, PCR, and cell viability analysis. Murine macrophage cell line, RAW264.7 cells were cultured and used to generate M1 MEVs for use in ovarian cancer xenograft models. Results: M1 MEVs can effectively convert M2 macrophages to an M1-like state both in isolation and when co-cultured with ovarian cancer cells in vitro, resulting in a reduced ovarian cancer cell viability. Additionally, RAW264.7 M1 MEVs can localize to ovarian cancer tumor xenografts in mice. Conclusion: Human M1 MEVs can repolarize M2 macrophages to a M1 state and have anti-cancer activity against ovarian cancer cell lines. RAW264.7 M1 MEVs localize to tumor xenografts in vivo murine models.

Highly homologous mouse Cyp2a4 and Cyp2a5 genes are differentially expressed in the liver and both express long non-coding antisense RNAs.

The mammalian Cytochrome P450 (Cyp) gene superfamily encodes enzymes involved in numerous metabolic pathways and are frequently expressed in the liver. Despite the remarkably high sequence similarity of Cyp2a4 and Cyp2a5 genes and their surrounding genomic regions, they exhibit differences in expression in the adult mouse liver. For example, Cyp2a4 is highly female-biased whereas Cyp2a5 is only moderately female-biased and Cyp2a4, but not Cyp2a5, is activated in liver cancer. We hypothesized that the limited sequence differences may help us identify the basis for this differential expression. An antisense expressed sequence tag had been uniquely annotated to the Cyp2a4 gene which led us to investigate this transcript as a possible regulator of this gene. We characterized the full-length antisense transcript and also discovered a similar transcript in the Cyp2a5 gene. These transcripts are nuclear long noncoding RNAs that are expressed similarly to their sense mRNA counterparts. This includes the sex-biased and liver tumor differences seen between the Cyp2a4 and Cyp2a5 genes, but we also find that these two genes and their antisense transcripts are expressed within different zones of the liver structure. Interestingly, while the differences in sex-biased expression of the mRNAs are established 1-2 months after birth, the antisense transcripts exhibit these expression differences earlier, at 3-4 weeks after birth. By analyzing published genomic data, we have identified candidate transcription factor binding sites that could account for differences in Cyp2a4/Cyp2a5 expression. Taken together, these studies characterize the first antisense RNAs within the Cyp supergene family and identify potential transcriptional and post-transcriptional mechanisms governing different Cyp2a4 and Cyp2a5 expression patterns in mouse liver.

Evolutionary Analysis of the Zinc Finger and Homeoboxes Family of Proteins Identifies Multiple Conserved Domains and a Common Early Chordate Ancestor.

The Zinc Fingers and Homeoboxes (Zhx) proteins, Zhx1, Zhx2, and Zhx3, comprise a small family of proteins containing two amino-terminal C2-H2 zinc fingers and four or five carboxy-terminal homeodomains. These multiple homeodomains make Zhx proteins unusual because the majority of homeodomain-containing proteins contain a single homeodomain. Studies in cultured cells and mice suggest that Zhx proteins can function as positive or negative transcriptional regulators. Zhx2 regulates numerous hepatic genes, and all three Zhx proteins have been implicated in different cancers. Because Zhx proteins contain multiple predicted homeodomains, are associated with interesting physiological traits, and seem to be only present in the vertebrate lineage, we investigated the evolutionary history of this small family by comparing Zhx homologs from a wide range of chordates. This analysis indicates that the zinc finger motifs and homeodomains are highly similar among all Zhx proteins and also identifies additional Zhx-specific conserved regions, including a 13 amino acid amino-terminal motif that is nearly identical among all gnathostome Zhx proteins. We found single Zhx proteins in the sea lamprey (Petromyzon marinus) and in the nonvertebrate chordates sea squirt (Ciona intestinalis) and lancelet (Branchiostoma floridae); these Zhx proteins are most similar to gnathostome Zhx3. Based on our analyses, we propose that a duplication of the primordial Zhx gene gave rise to Zhx3 and the precursor to Zhx1 and Zhx2. A subsequent tandem duplication of this precursor generated Zhx1 and Zhx2 found in gnathostomes.