Simultaneous down-regulation of tumor suppressor genes RBSP3/CTDSPL, NPRL2/G21 and RASSF1A in primary non-small cell lung cancer

<p>Abstract</p> <p>Background</p> <p>The short arm of human chromosome 3 is involved in the development of many cancers including lung cancer. Three bona fide lung cancer tumor suppressor genes namely <it>RBSP3 </it>(AP20 region),<it>NPRL2 </it>and <it>RASSF1A </it>(LUCA region) were identified in the 3p21.3 region. We have shown previously that homozygous deletions in AP20 and LUCA sub-regions often occurred in the same tumor (P < 10^-6).</p> <p>Methods</p> <p>We estimated the quantity of <it>RBSP3, NPRL2, RASSF1A, GAPDH, RPN1 </it>mRNA and <it>RBSP3 </it>DNA copy number in 59 primary non-small cell lung cancers, including 41 squamous cell and 18 adenocarcinomas by real-time reverse transcription-polymerase chain reaction based on TaqMan technology and relative quantification.</p> <p>Results</p> <p>We evaluated the relationship between mRNA level and clinicopathologic characteristics in non-small cell lung cancer. A significant expression decrease (≥2) was found for all three genes early in tumor development: in 85% of cases for <it>RBSP3</it>; 73% for <it>NPRL2 </it>and 67% for <it>RASSF1A </it>(P < 0.001), more strongly pronounced in squamous cell than in adenocarcinomas. Strong suppression of both, <it>NPRL2 </it>and <it>RBSP3 </it>was seen in 100% of cases already at Stage I of squamous cell carcinomas. Deregulation of <it>RASSF1A </it>correlated with tumor progression of squamous cell (P = 0.196) and adenocarcinomas (P < 0.05). Most likely, genetic and epigenetic mechanisms might be responsible for transcriptional inactivation of <it>RBSP3 </it>in non-small cell lung cancers as promoter methylation of <it>RBSP3 </it>according to NotI microarrays data was detected in 80% of squamous cell and in 38% of adenocarcinomas. With NotI microarrays we tested how often LUCA (<it>NPRL2, RASSF1A</it>) and AP20 (<it>RBSP3</it>) regions were deleted or methylated in the same tumor sample and found that this occured in 39% of all studied samples (P < 0.05).</p> <p>Conclusion</p> <p>Our data support the hypothesis that these TSG are involved in tumorigenesis of NSCLC. Both genetic and epigenetic mechanisms contribute to down-regulation of these three genes representing two tumor suppressor clusters in 3p21.3. Most importantly expression of <it>RBSP3, NPRL2 </it>and <it>RASSF1A </it>was simultaneously decreased in the same sample of primary NSCLC: in 39% of cases all these three genes showed reduced expression (P < 0.05).</p

Differential Expression of CHL1 Gene during Development of Major Human Cancers

CHL1 gene (also known as CALL) on 3p26.3 encodes a one-pass trans-membrane cell adhesion molecule (CAM). Previously CAMs of this type, including L1, were shown to be involved in cancer growth and metastasis.We used Clontech Cancer Profiling Arrays (19 different types of cancers, 395 samples) to analyze expression of the CHL1 gene. The results were further validated by RT-qPCR for breast, renal and lung cancer. Cancer Profiling Arrays revealed differential expression of the gene: down-regulation/silencing in a majority of primary tumors and up-regulation associated with invasive/metastatic growth. Frequent down-regulation (>40% of cases) was detected in 11 types of cancer (breast, kidney, rectum, colon, thyroid, stomach, skin, small intestine, bladder, vulva and pancreatic cancer) and frequent up-regulation (>40% of cases)--in 5 types (lung, ovary, uterus, liver and trachea) of cancer. Using real-time quantitative PCR (RT-qPCR) we found that CHL1 expression was decreased in 61% of breast, 60% of lung, 87% of clear cell and 89% papillary renal cancer specimens (P<0.03 for all the cases). There was a higher frequency of CHL1 mRNA decrease in lung squamous cell carcinoma compared to adenocarcinoma (81% vs. 38%, P = 0.02) without association with tumor progression.Our results suggested that CHL1 is involved in the development of different human cancers. Initially, during the primary tumor growth CHL1 could act as a putative tumor suppressor and is silenced to facilitate in situ tumor growth for 11 cancer types. We also suggested that re-expression of the gene on the edge of tumor mass might promote local invasive growth and enable further metastatic spread in ovary, colon and breast cancer. Our data also supported the role of CHL1 as a potentially novel specific biomarker in the early pathogenesis of two major histological types of renal cancer

Tumor suppressor function of the SEMA3B gene in human lung and renal cancers

The SEMA3B gene is located in the 3p21.3 LUCA region, which is frequently affected in different types of cancer. The objective of our study was to expand our knowledge of the SEMA3B gene as a tumor suppressor and the mechanisms of its inactivation. In this study, several experimental approaches were used: tumor growth analyses and apoptosis assays in vitro and in SCID mice, expression and methylation assays and other. With the use of the small cell lung cancer cell line U2020 we confirmed the function of SEMA3B as a tumor suppressor, and showed that the suppression can be realized through the induction of apoptosis and, possibly, associated with the inhibition of angiogenesis. In addition, for the first time, high methylation frequencies have been observed in both intronic (32-39%) and promoter (44-52%) CpG-islands in 38 non-small cell lung carcinomas, including 16 squamous cell carcinomas (SCC) and 22 adenocarcinomas (ADC), and in 83 clear cell renal cell carcinomas (ccRCC). Correlations between the methylation frequencies of the promoter and the intronic CpG-islands of SEMA3B with tumor stage and grade have been revealed for SCC, ADC and ccRCC. The association between the decrease of the SEMA3B mRNA level and hypermethylation of the promoter and the intronic CpG-islands has been estimated in renal primary tumors (P < 0.01). Using qPCR, we observed on the average 10- and 14-fold decrease of the SEMA3B mRNA level in SCC and ADC, respectively, and a 4-fold decrease in ccRCC. The frequency of this effect was high in both lung (92-95%) and renal (84%) tumor samples. Moreover, we showed a clear difference (P < 0.05) of the SEMA3B relative mRNA levels in ADC with and without lymph node metastases. We conclude that aberrant expression and methylation of SEMA3B could be suggested as markers of lung and renal cancer progression

High Mutability of the Tumor Suppressor Genes RASSF1 and RBSP3 (CTDSPL) in Cancer

BACKGROUND:Many different genetic alterations are observed in cancer cells. Individual cancer genes display point mutations such as base changes, insertions and deletions that initiate and promote cancer growth and spread. Somatic hypermutation is a powerful mechanism for generation of different mutations. It was shown previously that somatic hypermutability of proto-oncogenes can induce development of lymphomas. METHODOLOGY/PRINCIPAL FINDINGS:We found an exceptionally high incidence of single-base mutations in the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) both located in 3p21.3 regions, LUCA and AP20 respectively. These regions contain clusters of tumor suppressor genes involved in multiple cancer types such as lung, kidney, breast, cervical, head and neck, nasopharyngeal, prostate and other carcinomas. Altogether in 144 sequenced RASSF1A clones (exons 1-2), 129 mutations were detected (mutation frequency, MF = 0.23 per 100 bp) and in 98 clones of exons 3-5 we found 146 mutations (MF = 0.29). In 85 sequenced RBSP3 clones, 89 mutations were found (MF = 0.10). The mutations were not cytidine-specific, as would be expected from alterations generated by AID/APOBEC family enzymes, and appeared de novo during cell proliferation. They diminished the ability of corresponding transgenes to suppress cell and tumor growth implying a loss of function. These high levels of somatic mutations were found both in cancer biopsies and cancer cell lines. CONCLUSIONS/SIGNIFICANCE:This is the first report of high frequencies of somatic mutations in RASSF1 and RBSP3 in different cancers suggesting it may underlay the mutator phenotype of cancer. Somatic hypermutations in tumor suppressor genes involved in major human malignancies offer a novel insight in cancer development, progression and spread

Notl subtraction and Notl-specific microarrays to detect copy number and methylation changes in whole genomes

  Methylation, deletions, and amplifications of cancer genes constitute important mechanisms in carcinogenesis. For genome-wide analysis of these changes, Li et al propose the use of Notl clone microarrays and genomic subtraction, because Notl recognition sites are closely associated with CpG islands and genes

Tumor Suppressor Properties of Small C-Terminal Domain Phosphatases in Clear Cell Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC) accounts for 80–90% of kidney cancers worldwide. Small C-terminal domain phosphatases CTDSP1, CTDSP2, and CTDSPL (also known as SCP1, 2, 3) are involved in the regulation of several important pathways associated with carcinogenesis. In various cancer types, these phosphatases may demonstrate either antitumor or oncogenic activity. Tumor-suppressive activity of these phosphatases in kidney cancer has been shown previously, but in general case, the antitumor activity may be dependent on the choice of cell line. In the present work, transfection of the Caki-1 cell line (ccRCC morphologic phenotype) with expression constructs containing the coding regions of these genes resulted in inhibition of cell growth in vitro in the case of CTDSP1 (p CTDSPL (p CTDSP2. The analysis of The Cancer Genome Atlas (TCGA) data showed differential expression of some of CTDSP genes and of their target, RB1. These results were confirmed by quantitative RT-PCR using an independent sample of primary ccRCC tumors (n = 52). We observed CTDSPL downregulation and found a positive correlation of expression for two gene pairs: CTDSP1 and CTDSP2 (rs = 0.76; p CTDSPL and RB1 (rs = 0.38; p CTDSP1, CTDSP2, CTDSPL, and RB1 with poor survival of ccRCC patients (p CTDSP1, CTDSP2, and RB1 were differently expressed in two subtypes of ccRCC—ccA and ccB, characterized by different survival rates. These results confirm that CTDSP1 and CTDSPL have tumor suppressor properties in ccRCC and reflect their association with the more aggressive ccRCC phenotype

Технология переработки малосульфидной золото-кварцевой руды

Актуальность исследования обусловлена необходимостью получения новых знаний об обогатимости золотосодержащих руд и необходимостью развития ресурсной базы Российской Федерации, а именно вовлечения объектов недропользования в хозяйственный оборот. Цель: исследование по переработке малосульфидной золото-кварцевой руды с применением комплексной технологии обогащения минерального сырья. Объект: золотосодержащая руда со средним содержанием золота 11,88 г/т. Содержание серебра незначительное - 2,43 г/т. Основными рудными минералами в образце являются пирит и пирротин. Среднее содержание этих минералов в руде, по данным минералогического и рентгеноструктурного анализа, составило около 6 % (суммарно). Основными породообразующими минералами исходной руды являются: кварц - 60,1 %; кварц-хлорит-слюдяные агрегаты - 3,8 %; карбонаты - 7,1 %. Методы. Методология исследования базировалась на теоретических основах обогащения полезных ископаемых. Изучение вещественного состава руды и продуктов обогащения было выполнено с использованием химического, пробирного, термического, спектрометрического методов, а также атомно-эмиссионной спектрометрии и других методов. Изучение обогатимости проводили методами гравитационного и флотационного обогащения, а именно: GRG тест, стадиальный тест института ТОМС (Россия) (определение оптимальной крупности измельчения руды и количества стадий обогащения), моделирование извлечения золота в цикле измельчения, определение оптимальной степени помола, исследование выбора флотационных реагентов, исследование кинетики процесса и проведение комплекса технологических исследований. Результаты. Установлено, что извлечение золота при проведении GRG-теста составило 72,75 % при выходе общего концентрата 1,34 % и его содержании 664,78 г/т. При этом содержание золота в хвостах составило 3,29 г/т. Стадийные испытания показали, что при переработке руд только гравитационным способом целесообразно использовать двухстадийную схему. Первая стадия находится в цикле измельчения при крупности руды 60-70 % и вторая стадия - при конечной крупности насыпи классификации 90 % класса -0,071 мм. Центробежная сепарация как свободная операция извлечения золота в цикле измельчения работает эффективно. Получен концентрат с содержанием золота 2426 г/т с выходом 0,31 % и извлечением 63,74 %. Обогащение дробленых до 90 % хвостов первой очереди класса -0,071 мм на обогатительной фабрике KC-CVD (моделирование) позволило извлечь золото в общий гравиоконцентрат (KC-MD+KC-CVD) 87,25 % при содержании выхода фугата 22,63 %. Содержание золота в хвостах составило 1,97 г/т.The relevance of the research is caused by the necessity to acquire new knowledge about the enrichment of gold-bearing ores and the need to develop the resource base of the Russian Federation, namely involve subsoil use objects in economic circulation. The purpose of the study is to study the processing of low-sulfide gold-quartz ore using an integrated technology for the enrichment of mineral raw materials. Object: gold ore raw materials with an average gold grade of 11,88 g/t. The silver content is negligible - 2,43 g/t. The main ore minerals in the sample are pyrite and pyrrhotite. The average content of these minerals in the ore, according to mineralogical and X-ray diffraction analysis, was about 6 % (in total). The main rock-forming minerals of the original ore are: quartz - 60,1 %; quartz-chlorite-mica aggregates - 3,8 %; carbonates - 7,1 %. Methods. The research methodology was based on the theoretical foundations of mineral processing. The study of the material composition of the ore, and enrichment products, was carried out using chemical, assay, thermal, spectrometric methods, as well as atomic emission spectrometry and other methods. The study of washability was carried out by the methods of gravity and flotation enrichment, namely: GRG test, stage test of the Institute TOMS (Russia) (determination of the optimal size of ore grinding and the number of enrichment stages), modeling of gold extraction in the grinding cycle, determination of the optimal degree of grinding, study of the choice of flotation reagents, study of the kinetics of the process and the implementation of a complex of technological studies. Results. It was found that the gold recovery when performing the GRG test was 72,75 % with a total concentrate yield of 1,34 % and a content of Au 664,78 g/t. At the same time, the gold content in the tailings was 3,29 g/t. A stage test showed that for ore processing only by gravity technology, it is advisable to use a two-stage scheme. The first stage is in the grinding cycle with the ore size of 60-70 % and the second stage - with the final size of the discharge of the classification 90 % size of -0,071 mm. Centrifugal separation, as a free gold recovery operation in a grinding cycle, works efficiently. A concentrate with a gold content of 2426 g/t was obtained with an output of 0,31 % and an extraction of 63,74 %. The enrichment of the first stage tailings crushed to 90 % size of -0,071 mm at the KC-CVD concentrator (modeling) made it possible to extract gold into a total gravity concentrate (KC-MD+KC-CVD) of 87,25 % with a concentrate yield of 22,63 %. The gold content in the tailings was 1,97 g/t. The results of gravity and flotation concentration of the original ore indicate the feasibility of using a combined gravity-flotation technological scheme. In a closed experiment of the initial ore beneficiation according to the gravity-flotation scheme at a natural pH of the pulp (without adding acid), the following products were obtained: gravity concentrate with a gold content of 2426 g/t at a yield of 0,31 % and an extraction of 64,06 %; flotation concentrate (after the II cleaning) with a gold content of 122 g/t with a yield of 2,90 % and recovery of 33,01 %; the total gold recovery in the gravity-flotation concentrate was 94,07 % with a yield of 3,21 % and Au content of 345,87 g/t, the gold content in the flotation tailings was 0,72 g/t

SILENCING MICAL2 REPRESSES HUMAN CANCER CELL GROWTH AND INVASION INDUCING MESENCHYMAL TO EPITHELIAL TRANSITION

MICAL (Molecules Interacting with CasL)2 belongs to an evolutionarily conserved family of proteins that catalyze actin oxidation-reduction reactions destabilizing F-actin in cytoskeletal dynamics. Here we show for the first time that MICAL2 mRNA is significantly over-expressed in aggressive, poorly differentiated/ undifferentiated, primary gastric and renal human epithelial cancers. Immunohistochemistry showed MICAL2- positive cells on the cancer invasive front and in metastasizing cancer cells inside emboli, but not at sites of metastasis, suggesting MICAL2 expression was “on” in a subpopulation of primary cancer cells seemingly detaching from the tissue of origin, enter emboli and travel to distant sites, to be turned “off” once homing at the metastatic site occurred. In vitro, MICAL2 knock-down was clearly associated with induction of mesenchymal to epithelial transition, causing reduction of viability and loss of motility and invasion properties of human cancer cells. Moreover, expression of MICAL2 cDNA in MICAL2-depleted cells induced epithelial to mesenchymal transition. All together our data indicate MICAL2 over-expression is associated with cancer progression and metastatic disease. MICAL2 might be an important regulator of epithelial to mesenchymal transition and therefore a promising target for anti-metastatic therapy