Arvydas Kanopka

Taking advantage of tumor cell adaptations to hypoxia for developing new tumor markers and treatment strategies.

Cancer cells in hypoxic areas of solid tumors are to a large extent protected against the action of radiation as well as many chemotherapeutic drugs. There are, however, two different aspects of the problem caused by tumor hypoxia when cancer therapy is concerned: One is due to the chemical reactions that molecular oxygen enters into therapeutically targeted cells. This results in a direct chemical protection against therapy by the hypoxic microenvironment, which has little to do with cellular biological regulatory processes. This part of the protective effect of hypoxia has been known for more than half a century and has been studied extensively. However, in recent years there has been more focus on the other aspect of hypoxia, namely the effect of this microenvironmental condition on selecting cells with certain genetic prerequisites that are negative with respect to patient prognosis. There are adaptive mechanisms, where hypoxia induces regulatory cascades in cells resulting in a changed metabolism or changes in extracellular signaling. These processes may lead to changes in cellular intrinsic sensitivity to treatment irrespective of oxygenation and, furthermore, may also have consequences for tissue organization. Thus, the adaptive mechanisms induced by hypoxia itself may have a selective effect on cells, with a fine-tuned protection against damage and stress of many kinds. It therefore could be that the adaptive mechanisms may take advantage of for new tumor labeling/imaging and treatment strategies. One of the Achilles’ heels of hypoxia research has always been the exact measurements of tissue oxygenation as well as the control of oxygenation in biological tumor models. Thus, development of technology that can ease this control is vital in order to study mechanisms and perform drug development under relevant conditions. An integrated EU Framework project 2004–2009, termed EUROXY, demonstrates several pathways involved in transcription and translation control of the hypoxic cell phenotype and evidence of cross-talk with responses to pH and redox changes. The carbonic anhydrase isoenzyme CA IX was selected for further studies due to its expression on the surface of many types of hypoxic tumors. The effort has led to marketable culture flasks with sensors and incubation equipment, and the synthesis of new drug candidates against new molecular targets. New labeling/imaging methods for cancer diagnosing and imaging of hypoxic cancer tissue are now being tested in xenograft models and are also in early clinical testing, while new potential anti-cancer drugs are undergoing tests using xenografted tumor cancers. The present article describes the above results in individual consortium partner presentations.

Transcriptional up-regulation of inhibitory PAS domain protein gene expression by hypoxia-inducible factor 1 (HIF-1) : a negative feedback regulatory circuit in HIF-1-mediated signaling in hypoxic cells

The inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS), a dominant negative regulator of hypoxia-inducible transcription factors (HIFs), is potentially implicated in negative regulation of angiogenesis in such tissues as the avascular cornea of the eye. We have previously shown IPAS mRNA expression is up-regulated in hypoxic tissues, which at least in part involves hypoxia-dependent alternative splicing of the transcripts from the IPAS/HIF-3α locus. In the present study, we demonstrate that a hypoxia-driven transcriptional mechanism also plays a role in augmentation of IPAS gene expression. Isolation and analyses of the promoter region flanking to the first exon of IPAS gene revealed a functional hypoxia response element at position –834 to –799, whereas the sequence upstream of the HIF-3α first exon scarcely responded to hypoxic stimuli. A transient transfection experiment demonstrated that HIF-1α mediates IPAS promoter activation via the functional hypoxia response element under hypoxic conditions and that a constitutively active form of HIF-1α is sufficient for induction of the promoter in normoxic cells. Moreover, chromatin immunoprecipitation and electrophoretic mobility shift assays showed binding of the HIF-1 complex to the element in a hypoxia-dependent manner. Taken together, HIF-1 directly up-regulates IPAS gene expression through a mechanism distinct from RNA splicing, providing a further level of negative feedback gene regulation in adaptive responses to hypoxic/ischemic conditions.

G/A polymorphism in intronic sequence affects the processing of MAO-B gene in patients with Parkinson disease

Monoamine oxidase B (MAO‐B) plays an important role in the metabolism of neuroactive and vasoactive amines in the central nervous system and peripheral tissues. Increased levels of MAO‐B mRNA and enzymatic activity have been reported in platelets from patients with Parkinsons and Alzheimers diseases, however the triggers of enhanced mRNA levels are unknown. Our results demonstrate for the first time that G/A dimorphism in intron 13 sequence creates splicing enhancer thus stimulating intron 13 removal efficiency. The increased MAO‐B protein levels might serve as a surrogate marker for – Parkinson disease.

Increased Serine-Arginine (SR) Protein Phosphorylation Changes Pre-mRNA Splicing in Hypoxia

Background: Splicing machinery heavily contributes to the ability of cells to adapt to hypoxic conditions. Results: SR proteins become hyperphosphorylated in hypoxia by HIF-1-dependent increase in SR protein kinase expression. Conclusion: SR proteins are one of hypoxia-dependent pre-mRNA splicing regulators. Significance: This is the first elucidation of factors involved in hypoxia-dependent splicing regulation. The removal of introns from mRNA precursors (pre-mRNAs) is an essential step in eukaryotic gene expression. The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Inhibitory PAS domain protein (IPAS), a dominant negative regulator of hypoxia-inducible gene expression, is generated from hypoxia inducible transcription factor-3α (HIF-3α) pre-mRNA by an alternative splicing mechanism. Inactivation of the IPAS transcript in mice leads to the neo-vascularization of the cornea, suggesting that IPAS is an important regulator of anti-angiogenesis in this tissue. For the first time we demonstrate that serine-arginine (SR) proteins are involved in oxygen tension-dependent changes in pre-mRNA splicing. SR proteins isolated from hypoxic cells differentially interact with RNA (compared with proteins isolated from cells cultured under normoxic conditions). They possess the differential ability to activate hypoxia-dependent splice sites, and they are more phosphorylated than those isolated from normoxic HeLa cells. We also show that expression of SR protein kinases (CLK1, SRPK1, SRPK2) in hypoxic cells is elevated at mRNA and protein levels. Increased expression of CLK1 kinase is regulated by HIFs. Reduction of CLK1 cellular expression levels reduces hypoxia-dependent full-length carbonic anhydrase IX (CAIX) mRNA and CAIX protein formation and changes hypoxia-dependent cysteine-rich angiogenic inducer 61 (Cyr61) mRNA isoform formation profiles.

Discovery of two novel EWSR1/ATF1 transcripts in four chimerical transcripts-expressing clear cell sarcoma and their quantitative evaluation

The most common recurrent translocation in clear cell sarcoma t(12;22)(q13;q12) results in an EWSR1/ATF1 chimeric gene. We present a molecular analysis of tumor overgrowing right proximal tibia with bone destruction metastatic to two groin lymph nodes. Fluorescent in situ hybridization analysis performed on paraffin-embedded tissue sections of primary tumor sample indicated one rearranged locus of EWSR1 gene and one additional red signal. Reverse transcription-polymerase chain reaction analysis revealed the presence of four different EWSR1/ATF1 chimerical transcripts in the tumor sample as well as in both metastatic lymph nodes. Two previously described transcripts EWSR1exon7/ATF1exon5 and EWSR1exon8/ATF1exon4, and two novel transcripts EWSR1exon7/ATF1exon4 and EWSR1exon9/ATF1exon4 were identified. Both novel transcripts were out-of-frame fusions and, therefore, most likely had limited biological impact in oncogenesis of clear cell sarcoma. Quantitative evaluation demonstrated unequal distribution of these transcripts, with EWSR1exon8/ATF1exon4 type being overexpressed.

Splicing-dependent expression of microRNAs of mirtron origin in human digestive and excretory system cancer cells

BackgroundAn abundant class of intronic microRNAs (miRNAs) undergoes atypical Drosha-independent biogenesis in which the spliceosome governs the excision of hairpin miRNA precursors, called mirtrons. Although nearly 500 splicing-dependent miRNA candidates have been recently predicted via bioinformatic analysis of human RNA-Seq datasets, only a few of them have been experimentally validated. The detailed mechanism of miRNA processing by the splicing machinery and the roles of mirtronic miRNAs in cancer are yet to be uncovered.MethodsWe experimentally examined whether biogenesis of certain miRNAs is under a splicing control by analyzing their expression levels in response to alterations in the 5′- and 3′-splice sites of a series of intron-containing minigenes carrying appropriate miRNAs. The expression levels of the miRNAs processed from mirtrons were determined by quantitative real-time PCR in five digestive tract (pancreas PANC-1, SU.86.86, T3M4, stomach KATOIII, colon HCT116) and two excretory system (kidney CaKi-1, 786-O) carcinoma cell lines as well as in pancreatic, stomach, and colorectal tumors. Transiently expressed SRSF1 and SRSF2 splicing factors were quantified by western blotting in the nuclear fractions of HCT116 cells.ResultsWe found that biogenesis of the human hsa-miR-1227-3p, hsa-miR-1229-3p, and hsa-miR-1236-3p is splicing-dependent; therefore, these miRNAs can be assigned to the class of miRNAs processed by a non-canonical mirtron pathway. The expression analysis revealed a differential regulation of human mirtronic miRNAs in various cancer cell lines and tumors. In particular, hsa-miR-1229-3p is selectively upregulated in the pancreatic and stomach cancer cell lines derived from metastatic sites. Compared with the healthy controls, the expression of hsa-miR-1226-3p was significantly higher in stomach tumors but extensively downregulated in colorectal tumors. Furthermore, we provided evidence that overexpression of SRSF1 or SRSF2 can upregulate the processing of individual mirtronic miRNAs in HCT116 cells.ConclusionsAn interplay of different splicing factors, such as SRSF1 or SRSF2, may alter the levels of miRNAs of mirtron origin in a cell. Our findings underline the specific expression profiles of mirtronic miRNAs in colorectal, stomach, and pancreatic cancer.

Gastrointestinal tract tumors and cell lines possess differential splicing factor expression and tumor associated mRNA isoform formation profiles

BACKGROUND Cell lines derived from human tumors have been extensively used as experimental models of neoplastic disease. Although such cell lines differ from both normal and cancerous tissue. OBJECTIVE The data obtained used DNA and RNA microarray systems does not give full information about protein expression levels in cells and tissues. We present experimental evidence that splicing factor SRSF1, SRSF2, U2AF35, U2AF65 and KHSRP expression levels in gastrointestinal tract (colon, gastric and pancreatic) tumors differ compare to healthy tissues and in cell lines, derived from corresponding organs. METHODS Protein expression was analyzed using Western blots. RT-PCR method was used for Fas and Rac splicing analysis. RESULTS Obtained results provided a novel molecular characterization of this important group of human cell lines and their relationships to tumors in vivo. Expression levels of individual splicing factors in tumors might serve as tumor markers. Not all experimental results obtained from cell lines reflect changes that occur in tumors. Also Fas and Rac, cancer associated genes, tumor associated sFas and Rac1b mRNA isoform profiles in cell lines do not correspond to profiles that are observed in tumors. CONCLUSIONS Not all experimental results obtained in cell lines reflect changes that occur in real tumors.

Cell survival: Interplay between hypoxia and pre-mRNA splicing

RNA splicing takes place in the nucleus and occurs either co- or post-transcriptionally. Noncoding sequences (introns) in nuclear mRNA precursors (pre-mRNA) are removed by dedicated splicing machinery. The coding sequences (exons) are joined to generate the mature mRNA that is exported to the cytoplasm and translated into protein. Splicing events are tissue-specific. This process plays an important role in cellular differentiation and organism development. The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to different developmental stages and altered cellular conditions. A striking change has been observed in alternative splicing pattern of genes and alterations in splicing factor expression under pathologic conditions especially in human cancers. Cancer cells are often confronted with a significant reduction in oxygen availability, which is a major reason for changeover of major cellular processes. Hypoxic regions have been identified within all solid tumors and their presence has been linked to malignant progression, metastasis, resistance to therapy, and poor clinical outcomes following treatment. Cellular responses to hypoxia are mediated by hypoxia inducible transcription factors (HIFs). This review focuses on currently available data how pre-mRNAs splicing contributes to cellular adaptation to hypoxic conditions, to genes which alternative splicing is regulated dependent on hypoxia and how regulation of alternative splicing under hypoxic conditions is achieved.