Jana Jandova

Changes in mitochondrial DNA alter expression of nuclear encoded genes associated with tumorigenesis

We previously reported the presence of a mtDNA mutation hotspot in UV-induced premalignant and malignant skin tumors in hairless mice. We have modeled this change (9821insA) in murine cybrid cells and demonstrated that this alteration in mtDNA associated with mtBALB haplotype can alter the biochemical characteristics of cybrids and subsequently can contribute to significant changes in their behavioral capabilities. This study shows that changes in mtDNA can produce differences in expression levels of specific nuclear-encoded genes, which are capable of triggering the phenotypes such as seen in malignant cells. From a potential list of differentially expressed genes discovered by microarray analysis, we selected MMP-9 and Col1a1 for further studies. Real-time PCR confirmed up-regulation of MMP-9 and down-regulation of Col1a1 in cybrids harboring the mtDNA associated with the skin tumors. These cybrids also showed significantly increased migration and invasion abilities compared to wild type. The non-specific MMP inhibitor, GM6001, was able to inhibit migratory and invasive abilities of the 9821insA cybrids confirming a critical role of MMPs in cellular motility. Nuclear factor-κB (NF-κB) is a key transcription factor for production of MMPs. An inhibitor of NF-κB activation, Bay 11-7082, was able to inhibit the expression of MMP-9 and ultimately decrease migration and invasion of mutant cybrids containing 9821insA. These studies confirm a role of NF-κB in the regulation of MMP-9 expression and through this regulation modulates the migratory and invasive capabilities of cybrids with mutant mtDNA. Enhanced migration and invasion abilities caused by up-regulated MMP-9 may contribute to the tumorigenic phenotypic characteristics of mutant cybrids.

The matrix protein CCN1/CYR61 is required for αVβ5-mediated cancer cell migration

CYR61 is one of the six proteins of the CCN family of proteins known to play diverse roles in angiogenesis, cellular proliferation, survival, migration and wound healing. However, the specific function of CYR61 in cancer is unclear, and the literature remains controversial. We used quantitative real‐time PCR to establish the expression profile of CYR61 and integrin αVβ5 in three non–small cell lung cancer, five colorectal cancer, one breast cancer and one oesophageal squamous carcinoma cell lines. We showed that the levels of CYR61 were significantly increased in oesophageal squamous carcinoma cell line along with the enhanced levels of αVβ5 integrin. Further, we investigated whether tumour cell–secreted CYR61 can facilitate cell migration by interacting with the αVβ5 integrin. Using tumour cell lines with low, intermediate and high CYR61 expression and their isogenic variants as a cellular model, we determined that integrin αVβ5 expressed on these tumour cells is required for cell migration. Moreover, we showed that the modulation of expression levels of CYR61 in these cancer cells affected their capacity for migration. These results represent an advance to the understanding of the role of CYR61 and αvβ5 integrin as proteins that cooperate to mediate cancer cell migration. Copyright

The S100P/RAGE signaling pathway regulates expression of microRNA-21 in colon cancer cells

S100P signaling through the receptor for advanced glycation end‐products (RAGE) contributes to colon cancer invasion and metastasis, but the mechanistic features of this process are obscure. Here, we investigate whether activation of S100P/RAGE signaling regulates oncogenic microRNA‐21 (miR‐21). We show that exogenous S100P up‐regulates miR‐21 levels in human colon cancer cells, whereas knockdown of S100P results in a decrease of miR‐21. Furthermore, blockage of RAGE with anti‐RAGE antibody suppresses S100P induction of miR‐21. In addition, we found that S100P induction of miR‐21 expression involves ERK and is suppressed by the MEK inhibitor U0126. Also, S100P treatment stimulates the enrichment of c‐Fos, and AP‐1 family members, at the miR‐21 gene promoter.

Somatic alterations in mitochondrial DNA produce changes in cell growth and metabolism supporting a tumorigenic phenotype

There have been many reports of mitochondrial DNA (mtDNA) mutations associated with human malignancies. We have observed allelic instability in UV-induced cutaneous tumors at the mt-Tr locus encoding the mitochondrial tRNA for arginine. We examined the effects of somatic alterations at this locus by modeling the change in a uniform nuclear background by generating cybrids harboring allelic variation at mt-Tr. We utilized the naturally occurring mtDNA variation at mt-Tr within the BALB/cJ (BALB) and C57BL/6J (B6) strains of Mus musculus to transfer their mitochondria into a mouse ρ(0) cell line that lacked its own mtDNA. The BALB haplotype containing the mt-Tr 9821insA allele produced significant changes in cellular respiration (resulting in lowered ATP production), but increased rates of cellular proliferation in cybrid cells. Furthermore, the mtDNA genotype associated with UV-induced tumors endowed the cybrid cells with a phenotype of resistance to UV-induced apoptosis and enhanced migration and invasion capabilities. These studies support a role for mtDNA changes in cancer.

Identification of an mtDNA Mutation Hot Spot in UV-Induced Mouse Skin Tumors Producing Altered Cellular Biochemistry

There is increasing awareness of a role of mtDNA alterations in the development of cancer since mtDNA point mutations are found at high frequency in a variety of human tumors. To determine the biological effects of mtDNA mutations in UV-induced skin tumors, hairless mice were irradiated to produce tumors and the tumor mtDNAs were screened for single nucleotide changes using temperature gradient capillary electrophoresis (TGCE) followed by direct sequencing. A mutation hot spot (9821insA) in mt-Tr locus (tRNAArg) was discovered in approximately one third of premalignant and malignant skin tumors. To determine the functional relevance of this particular mutation in vitro, cybrid cell lines containing different mt-Tr (tRNAArg) alleles were generated. The resulting cybrid cell lines contain the same nuclear genotype and differ only in their mtDNA. The biochemical analysis of the cybrids revealed that the mutant haplotype is associated with diminished levels of complex I protein resulting in lower levels of baseline oxygen consumption and lower cellular ATP production. We hypothesize that this specific mtDNA mutation alters cellular biochemistry supporting the development of keratinocyte neoplasia.

Cyclophilin 40 alters UVA-induced apoptosis and mitochondrial ROS generation in keratinocytes.

Abstract The CyP40 protein encoded by PPID gene is a member of the peptidyl-prolyl cis–trans isomerase (PPIase) family. PPIases catalyze the cis–trans isomerization of proline imidic peptide bonds in oligopeptides and accelerate the folding of proteins. The CyP40 protein has been shown to possess PPIase activity and, similar to other family members, can bind to the immunosuppressant drug cyclosporin A (CsA). In this study, we created keratinocyte cell lines with CyP40 being stably knocked down using viral particles containing shRNA for CyP40 which knocked down the expression level of CyP40 transcripts by 90–99%. The proliferation rates of the cell lines with silenced CyP40 were decreased compared to the control cells. After UVA irradiation, the rate of apoptosis was found to be significantly lower in CyP40 silenced cell lines than it was in control cells. Moreover, mitochondrial membrane potential (MMP) was found to be less dissipated and mitochondrial permeability transition pore (MPTP) less active in cells with knocked down CyP40 than in control cells after UVA irradiation. Also, less mitochondrial superoxide was detected in the cells with silenced CyP40 compared to control cells after UVA exposure. Moreover, silencing of CyP40 partially modulates expression of key genes involved in mitochondrial pore formation including CyPD, ANTs and VDAC family members. The ability of CyP40 to regulate UV induced apoptosis implicates this protein as a potential target for therapy in cancer cells.

Modulation of ROS levels in fibroblasts by altering mitochondria regulates the process of wound healing

Mitochondria are the major source of reactive oxygen species (ROS) in fibroblasts which are thought to be crucial regulators of wound healing with a potential to affect the expression of nuclear genes involved in this process. ROS generated by mitochondria are involved in all stages of tissue repair process but the regulation of ROS-generating system in fibroblasts still remains poorly understood. The purpose of this study was to better understand molecular mechanisms of how the regulation of ROS levels generated by mitochondria may influence the process of wound repair. Cybrid model system of mtDNA variations was used to study the functional consequences of altered ROS levels on wound healing responses in a uniform nuclear background of cultured ρ0 fibroblasts. Mitochondrial ROS in cybrids were modulated by antioxidants that quench ROS to examine their ability to close the wound. Real-time PCR arrays were used to investigate whether ROS generated by specific mtDNA variants have the ability to alter expression of some key nuclear-encoded genes central to the wound healing response and oxidative stress. Our data suggest levels of mitochondrial ROS affect expression of some nuclear encoded genes central to wound healing response and oxidative stress and modulation of mitochondrial ROS by antioxidants positively affects in vitro process of wound closure. Thus, regulation of mitochondrial ROS-generating system in fibroblasts can be used as effective natural redox-based strategy to help treat non-healing wounds.

Disparities in incidence of early- and late-onset colorectal cancer between Hispanics and Whites: A 10-year SEER database study ☆

BACKGROUND Racial disparities in incidence of colorectal cancer (CRC) exist. In Hispanics, CRC was the second most commonly diagnosed cancer in 2012. METHODS We abstracted the national estimates for Hispanics/Whites with CRC using the SEER database between 2000 and 2010. Trends in incidence, mortality, gender and stage of disease were analyzed for early-onset (age<50; EO - young) and late-onset (age>50; LO - old) cases. RESULTS The overall incidence of CRC increased by 48% in Hispanics. 38% increase in incidence of LO CRC and 80% increase in incidence of EO CRC was seen in this ethnic group. Hispanics and Whites showed higher percentage of distant tumors for both age groups. There was no deviation in overall trend between males and females. CONCLUSIONS Although there is an overall decrease in incidence of CRC in Whites increase was seen in Hispanics. While incidence of EO CRC is increasing in both races, LO CRC incidence is increasing in Hispanics not in Whites. This data suggest that disparities in incidence of EO and LO CRC exist between Hispanics and Whites.

Sporadic early-onset colon cancer expresses unique molecular features

BACKGROUND The overall incidence of colon cancer (CC) has steadily declined in the last decades but has increased in patients under age 50 y. The etiology of early-onset (EO) CC is not understood. The aim of this study was to elucidate gene expression patterns in EOCC and show its uniqueness compared to late-onset (LO) disease. METHODS Two cohorts of patients with sporadic CC were identified. Tumors and matching noninvolved tissues from six EOCC patients (<50) and six late-onset colon cancers (LOCC) patients (>65) were obtained from pathology archives. De-paraffinized tissues were macrodissected from FFPE sections, RNA isolated, and used for expression profiling of 770 cancer-related genes representing 13 canonical pathways. RESULTS Among 770 genes assayed, changes in expression levels of 93 genes were statistically significant between EOCC and matching noninvolved tissues. There were also significant differences in expression levels of 118 genes between LOCC and matching noninvolved tissues. Detailed comparative gene expression analysis between EOCC and LOCC normalized to their matching noninvolved tissues revealed that changes in expression of 88 genes were unique to EOCC using the cutoff criteria of expression levels difference >2 fold and P value <0.01. From these differentially expressed genes specific to EOCC, 28 genes were upregulated and 60 genes downregulated. At the pathway level, RAS, MAPK, WNT, and DNARepair pathways were similarly deregulated in both age groups, whereas PI3K-AKT signaling was more specific to EOCC and cell cycle pathway to LOCC. CONCLUSIONS These results suggest that sporadic EOCC is characterized by distinct molecular events compared to LOCC.

Racial disparities and socioeconomic status in the incidence of colorectal cancer in Arizona

BACKGROUND The prevalence of racial and socioeconomic disparities in the development of colorectal cancer (CRC) is well known; however, statewide variability exits across the United States. The aim of our study was to determine the overall incidence, socioeconomic and racial disparities in the development of CRC in the state of Arizona. METHODS We performed a 16-year (1995 to 2011) retrospective review of the Arizona Cancer Registry including all patients with CRC. Patient demographics, stage of CRC disease, and patient outcomes were recorded. The outcome measures were incidence of CRC and the difference in racial and economic characteristics among patients. Logistic regression analysis was performed to identify factors associated with the incidence of CRC. RESULTS A total of 40,314 patients with CRC were included of which 16% (n = 6,450) were stage IV. The overall incidence of CRC decreased 17% over the study period. The highest incidence rates were seen in White non-Hispanic and African American populations. Right-sided tumors were more common in White non-Hispanic and African Americans whereas American Indians had higher incidence of rectal tumors and Asian/Pacific Islanders more commonly had left-sided tumors. African Americans had the highest occurrence (42.8%) of more advanced disease (stage III and stage IV). A negative correlation existed between socioeconomic status and the incidence of CRC. CONCLUSIONS Overall CRC incidence decreased in Arizona by 17%, with greatest decrease rate among, White non-Hispanic and African American populations. Educated patients with higher economic earnings experienced a lower decrease in the incidence of CRC.