Isoforms In Cancer Research Articles

Abstract IA13: Targeting PI3K isoforms in cancer: From mouse genetics to human therapy

Abstract The signaling axis of the class-I phosphatidylinositol 3-kinase (PI3K) is perhaps most commonly activated in cancer; activation of this pathway occurs via distinct mechanisms that include somatic activating mutations in PIK3CA (the gene encoding the p110α isoform of PI3K), genetic or epigenetic loss of the PTEN tumor suppressor and mutation/overexpression of RTKs. Thus PI3K represents an attractive target for cancer therapy and tremendous efforts are being devoted to developing effective PI3K inhibitors for cancer treatment. There are several PI3K isoforms in mammals, only the p110α and p110β isoforms are ubiquitously expressed. While the majority of the signal downstream of nearly all the RTKs and oncogenes so far tested is carried through p110α, p110β appears to play a prominent role in G protein mediated signaling in cells and for RTK signaling in certain circumstances. Surprisingly, we found that p110β is also the major player in a number of tumors driven by PTEN loss. In the mouse, each enzyme has been found to be necessary for certain processes and dispensable for others. Taking both genetic and pharmacological approaches, we have found that the two isoforms can actually play opposing roles in the normal development and pathology of mammary tissue. More recently, we found that p110α and p110β isoforms play spatially distinct roles in the epidermal skin that are required for the development and maintenance of PTEN hamartoma tumor syndrome. In this talk, I will discuss our most recent results on our studies of the distinct roles of the two isoforms in cancer. Citation Format: Jean J. Zhao. Targeting PI3K isoforms in cancer: From mouse genetics to human therapy. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA13.

Abstract IA12: New roles for PI3K isoforms in cancer

Abstract The PI3K pathway has been implicated in a variety of physiological responses, and is a therapeutic target in cancer and inflammation. Mammals have 8 distinct isoforms of PI3K. In order to gain insight into the physiological roles of PI3K isoforms, we have created ‘kinase knockin’ mice with germline inactivating or activating mutations in PI3K isoforms. This strategy more faithfully mimics pharmacological inhibitors or oncogenic activation than the classical KO/transgenic approaches, and has allowed us to uncover isoform-selective roles of class I isoforms of PI3K. An update on these efforts will be provided, uncovering new potential roles for PI3K isoforms in cancer therapy. Citation Format: Bart Vanhaesebroeck. New roles for PI3K isoforms in cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA12.

Expression modulation of different S6Ks isoforms in prostate cancer cell lines leads to alterations in proliferation, migration and chemotherapy resistance.

The mTOR pathway is stimulated by high amounts of amino acids, ATP, and insulin and several studies have shown that mTOR is active in various biochemical processes involved in cell growth and metabolism. The S6Ks proteins have shown an important role in the mTOR signaling, acting as effectors of this pathway. Many works have demonstrated the participation of the mTOR pathway in cancer, but there is currently a demand to better characterize the S6Ks isoforms in this disease. The present work aimed to study the roles of p70‐S6K1, p85‐S6K1 and p54‐S6K2 isoforms in cancer, using DU145 and PC3 prostate cancer cell lines. Our results indicate that p85‐S6K1 isoform enhances proliferation and colony formation of DU145 and PC3 cells. Besides, overexpression of both S6K1 isoforms increased migration of PC3 cells in scratch assay. Moreover, all S6Ks isoforms when overexpressed increased docetaxel resistance of PC3 cells. Finally, knockdown experiments against S6K1 and S6K2 had complemented our data so far. In conclusion, we have identified different cellular effects of S6Ks isoforms in an in vitro model of prostate cancer.

NADPH oxidases and cancer.

The mechanism by which reactive oxygen species (ROS) are produced by tumour cells remained incompletely understood until the discovery over the last 15 years of the family of NADPH oxidases (NOXs 1-5 and dual oxidases DUOX1/2) which are structural homologues of gp91phox, the major membrane-bound component of the respiratory burst oxidase of leucocytes. Knowledge of the roles of the NOX isoforms in cancer is rapidly expanding. Recent evidence suggests that both NOX1 and DUOX2 species produce ROS in the gastrointestinal tract as a result of chronic inflammatory stress; cytokine induction (by interferon-γ, tumour necrosis factor α, and interleukins IL-4 and IL-13) of NOX1 and DUOX2 may contribute to the development of colorectal and pancreatic carcinomas in patients with inflammatory bowel disease and chronic pancreatitis, respectively. NOX4 expression is increased in pre-malignant fibrotic states which may lead to carcinomas of the lung and liver. NOX5 is highly expressed in malignant melanomas, prostate cancer and Barrett's oesophagus-associated adenocarcinomas, and in the last it is related to chronic gastro-oesophageal reflux and inflammation. Over-expression of functional NOX proteins in many tissues helps to explain tissue injury and DNA damage from ROS that accompany pre-malignant conditions, as well as elucidating the potential mechanisms of NOX-related damage that contribute to both the initiation and the progression of a wide range of solid and haematopoietic malignancies.

Differential reprogramming of isogenic colorectal cancer cells by distinct activating KRAS mutations.

Oncogenic mutations of Ras at codons 12, 13, or 61, that render the protein constitutively active, are found in ∼16% of all cancer cases. Among the three major Ras isoforms, KRAS is the most frequently mutated isoform in cancer. Each Ras isoform and tumor type displays a distinct pattern of codon-specific mutations. In colon cancer, KRAS is typically mutated at codon 12, but a significant fraction of patients have mutations at codon 13. Clinical data suggest different outcomes and responsiveness to treatment between these two groups. To investigate the differential effects upon cell status associated with KRAS mutations we performed a quantitative analysis of the proteome and phosphoproteome of isogenic SW48 colon cancer cell lines in which one allele of the endogenous gene has been edited to harbor specific KRAS mutations (G12V, G12D, or G13D). Each mutation generates a distinct signature, with the most variability seen between G13D and the codon 12 KRAS mutants. One notable example of specific up-regulation in KRAS codon 12 mutant SW48 cells is provided by the short form of the colon cancer stem cell marker doublecortin-like Kinase 1 (DCLK1) that can be reversed by suppression of KRAS.

Abstract 717: Novel potent antisense oligonucleotides targeting transforming growth factor beta1 (TGF-β1)

Abstract Transforming growth factor beta (TGF-β) is a key member of a large family of cytokines, which play critical, pleiotropic roles in the pathophysiology of various human diseases, such as cancer, inflammation, autoimmune disease, and cirrhosis/fibrosis. TGF-β1, -2 and -3 isoforms are cytokines encoded by different genes but sharing strong sequence and structure homology. They function as the primary mediators of TGF-β signaling via both non-canonical and canonical signaling pathways. In Oncology, TGF-β isoforms are associated with a wide range of biological processes such as tumor cell invasion and migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Hence, blocking the TGF-β signaling pathway may have a multifold therapeutic benefit in Oncology, although therapeutic relevance of the respective TGF-β isoforms remains poorly documented. In order to evaluate the specific biological relevance of TGF-β1 isoform in cancer, we have initiated an extensive discovery program for identification of antisense oligodeoxynucleotide constructs selectively inhibiting expression of the TGF-β1 ligand. Based on the sequence of the human TGF-β1 mRNA, more than 150 Locked Nucleic Acid (LNA)-modified gapmers were designed, synthesized and tested in cell-based assays. Highly potent and selective TGF-β1 constructs were identified and selected based on efficient suppression of TGF-β1 mRNA/protein expression in different human and rodent tumor cell lines, and in human Peripheral Blood Mononuclear Cells (PBMCs). Effective target downregulation was demonstrated after lipofectamine aided-transfection (sub-nM concentration range), but also in the absence of any transfection agent (gymnotic delivery) at sub-µM concentrations. Selected TGF-β1 specific oligonucleotides were further tested in relevant animal xenograft models and strong target inhibition was also observed following systemic administration. Further details of this discovery program will be discussed and pharmacology properties and features of potent and selective TGF-β1 antagonists will be presented. Citation Format: Frank Jaschinski, Hanna Korhonen, Stephan Braun, Katja Wosikowski, Michel Janicot. Novel potent antisense oligonucleotides targeting transforming growth factor beta1 (TGF-β1). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 717. doi:10.1158/1538-7445.AM2014-717

CXCL12-γ in primary tumors drives breast cancer metastasis.

Compelling evidence shows that chemokine CXCL12 drives metastasis in multiple malignancies. Similar to other key cytokines in cancer, CXCL12 exists as several isoforms with distinct biophysical properties that may alter signaling and functional outputs. However, effects of CXCL12 isoforms in cancer remain unknown. CXCL12-α, β, and γ showed cell-type specific differences in activating signaling through G protein-dependent pathways in cell-based assays, while CXCL12-γ had greatest effects on recruitment of the adapter protein β-arrestin 2. CXCL12-β and γ also stimulated endothelial tube formation to a greater extent than CXCL12-α. To investigate effects of CXCL12 isoforms on tumor growth and metastasis, we used a mouse xenograft model of metastatic human breast cancer combining CXCR4+ breast cancer cells and mammary fibroblasts secreting an isoform of CXCL12. While all CXCL12 isoforms produced comparable growth of mammary tumors, CXCL12-γ significantly increased metastasis to bone marrow and other sites. Breast cancer cells originating from tumors with CXCL12-γ fibroblasts upregulated RANKL, contributing to bone marrow tropism of metastatic cancer cells. CXCL12-γ was expressed in metastatic tissues in mice, and we also detected CXCL12-γ in malignant pleural effusions from patients with breast cancer. In our mouse model, mammary fibroblasts disseminated to sites of breast cancer metastases, providing another mechanism to increase levels of CXCL12 in metastatic environments. These studies identify CXCL12-γ as a potent pro-metastatic molecule with important implications for cancer biology and effective therapeutic targeting of CXCL12 pathways.

Prognostic significance of the estrogen receptor beta (ERβ) isoforms ERβ1, ERβ2, and ERβ5 in advanced serous ovarian cancer

ObjectiveIn the present study we have examined the pattern of expression of the full length estrogen receptor β (ERβ1) and two ERβ splice variant isoforms (ERβ2, ERβ5) in well-characterized advanced serous ovarian cancers. MethodsImmunohistochemistry was performed with ERβ1, ERβ2, and ERβ5 antibodies and results were correlated with pathological and clinical follow-up data. Expression of ERβ isoforms in a panel of ovarian cancer cell lines and human tumor xenografts was also assessed. ResultsImmunohistochemical staining revealed cellular compartment-specific distribution for each isoform in malignant ovarian tissues exhibiting both nuclear staining and cytoplasmic staining. Patients with cytoplasmic ERβ2 expression had significantly worse outcome (p=0.006 at the multivariate analysis), the 5-year survival rate being nearly 28% for patients who did express cytoplasmic ERβ2, and 60% in negative patients. Cytoplasmic ERβ2 expression was also found to be significantly associated with chemoresistance. In concordance with clinical results both nuclear and cytoplasmic expressions were observed for the three isoforms in the cancer cell lines and human tumor xenografts tested. ConclusionsThis is the first study to uncover an unfavorable prognostic role of ERβ2 in advanced serous ovarian cancer. If anomalies of ERβ2 cytoplasmic expression could be demonstrated to represent an independent unfavorable prognostic marker and/or a marker predicting chemoresistance in advanced serous ovarian cancer, its immunohistochemical assessment at the time of surgery, could help to recognize candidates for clinical trials of new interventions.

Uncovering the role of p53 splice variants in human malignancy: a clinical perspective

Thirty-five years of research on p53 gave rise to more than 68,000 articles and reviews, but did not allow the uncovering of all the mysteries that this major tumor suppressor holds. How p53 handles the different signals to decide the appropriate cell fate in response to a stress and its implication in tumorigenesis and cancer progression remains unclear. Nevertheless, the uncovering of p53 isoforms has opened new perspectives in the cancer research field. Indeed, the human TP53 gene encodes not only one but at least twelve p53 protein isoforms, which are produced in normal tissues through alternative initiation of translation, usage of alternative promoters, and alternative splicing. In recent years, it became obvious that the different p53 isoforms play an important role in regulating cell fate in response to different stresses in normal cells by differentially regulating gene expression. In cancer cells, abnormal expression of p53 isoforms contributes actively to cancer formation and progression, regardless of TP53 mutation status. They can also be associated with response to treatment, depending on the cell context. The determination of p53 isoform expression and p53 mutation status helps to define different subtypes within a particular cancer type, which would have different responses to treatment. Thus, the understanding of the regulation of p53 isoform expression and their biological activities in relation to the cellular context would constitute an important step toward the improvement of the diagnostic, prognostic, and predictive values of p53 in cancer treatment. This review aims to summarize the involvement of p53 isoforms in cancer and to highlight novel potential therapeutic targets.

VEGF-A 165 family of isoforms as predictive biomarkers in patients with nonsquamous non-small cell lung cancer (NSCLC) treated with bevacizumab.

e19109 Background: Bevacizumab is a recombinant monoclonal humanized antibody to vascular endothelial growth factor (VEGF) that improves Time to Progression (TTP) in patients with advanced non-squamous NSCLC in combination with a doublet of platins, but currently no proven predictive markers exist. The VEGF-A 165 splice variant has been described as the most abundant and active isoform in cancer. Exon 8 splice modifications of VEGF 165 generates the VEGF-A 165a family of isoforms, which has a pro-angiogenic effect, and VEGF-A 165b family, with an anti-angiogenic activity. This study is aimed to explore the role of VEGF165a and VEGF165b expression in tumors as predictive biomarkers of efficacy in patients with NSCLC treated with platins plus bevacizumab. Methods: 22 patients were included (20 adenocarcinomas and 2 large cell carcinomas): 5 received carboplatin-taxol-bevacizumab, 14 carboplatin-taxotere-bevacizumab and 3 cisplatin-gemcitabine-bevacizumab. Total RNA was isolated by RNeasy FFPE procedure. VEGF165a and VEGF165b expression was analyzed by RT-qPCR using appropriate specific primers and probes. Individual VEGF165a and VEGF165b family of isoforms expression was calibrated to normal tissue and the ratio between both isoforms was calculated. Results: VEGF165a overexpression was detected in 14 (63.6%) cases and VEGF165b overexpression in 15 (68.2%). Individual overexpression for each family of isoforms was not predictive of benefit to bevacizumab therapy (p=0.933 and 0.166). However, the ratio between VEGF165a and VEGF165b was associated with TTP, correlating a predominant expression of pro-angiogenic VEGF165a with a significant benefit compared with cases with predominant VEGF165b expression (median TTP, 15 vs. 8 months respectively, p=0.005). The expression of both isoforms did not impact on OS (p=0.477). Conclusions: The overexpression of VEGF165a and low expression of VEGF165b family of isoforms correlated with benefit to anti-angiogenic therapy in NSCLC patients, supporting a potential use as predictive biomarkers for bevacizumab treatment in stage IV non-squamous NSCLC.

A requirement for wild-type Ras isoforms in mutant KRas-driven signalling and transformation

The mutant forms of KRas, NRas and HRas drive the initiation and progression of a number of human cancers, but less is known about the role of WT (wild-type) Ras alleles and isoforms in cancer. We used zinc-finger nucleases targeting HRas and NRas to modify both alleles of these genes in the mutant KRas-driven Hec1A endometrial cancer cell line, which normally expresses WT copies of these genes. The disruption of either WT isoform of Ras compromised growth-factor-dependent signalling through the ERK (extracellular-signal-regulated kinase) pathway. In addition, the disruption of HRas hindered the activation of Akt and subsequent downstream signalling. This was associated with decreased proliferation, increased apoptosis and decreased anchorage-independent growth in the HRas-disrupted cells. However, xenograft tumour growth was not significantly affected by the disruption of either NRas or HRas. As expected, deleting the mutant allele of KRas abolished tumour growth, whereas deletion of the remaining WT copy of KRas increased the tumorigenic properties of these cells; deleting a single copy of either HRas or NRas did not mimic this effect. The present study demonstrates that the WT copies of HRas, NRas and KRas play unique roles in the context of mutant KRas-driven tumours.

Functions, divergence and clinical value of TAp73 isoforms in cancer

The p73 gene encodes the tumour suppressive full-length TAp73 and N-terminal-truncated DNp73 isoforms that act as dominant negative inhibitors of TAp73. The overall effect of p73 in oncogenesis is thought to depend on the TAp73 to DNp73 isoforms' ratio. TAp73 isoforms include a number of C-terminal variants as a result of alternative splicing in 3'-end. TAp73 isoforms protect cells from oncogenic alterations in a multifaceted way since they are implicated in the suppression of all demonstrated hallmarks and enabling characteristics of cancer. Their best established role is in apoptosis, a process which seems to be differently affected by each TAp73 C-terminal variant. Based on previous findings and our thorough bioinformatics analysis, we highlight that TAp73 variants are functionally non-equivalent, since they present major differences in their transactivation efficiencies, protein interactions, response to DNA damage and apoptotic effects that are attributable to the primary structure of their C terminus. In this review, we summarise these differences and we unveil the link between crucial C-terminal motifs/residues and the oncosuppressive potential of TAp73 isoforms, emphasising on the importance of considering C terminus during the development of p73-based anticancer biologics.

Abstract 4618: Elucidating the role of distinct glutaminase isoforms in cancer.

Abstract Glutaminase is a key enzyme for the conversion of glutamine to glutamate with subsequent use as a fuel in the TCA cycle, glutathione production and protein synthesis. A subset of tumor cells shows preferential dependency on glutamine and glutaminase for cell proliferation and survival, consistent with “metabolic addiction” to this pathway. Between two glutaminase loci in the genome, the gene for kidney-type glutaminase (Gls1) has 2 alternatively spliced products (KGA and GAC), with distinct regulation and expression from liver-type glutaminase (Gls2). Furthermore, based on distinct C-termini and enzymatic properties, it has been proposed that the Gls1 alternatively spliced isoforms (KGA and GAC) may themselves have distinct functions. Our data further elucidates the role of glutaminase in cancer by addressing the following key questions: a) what is the major role of glutaminase in cells most sensitive to its inhibition? b) is GAC interchangeable with KGA in its expression pattern and role in tumor cells? c) how do tumors adapt to glutaminase inhibition? By generating and using a panel of GAC and KGA isoform-selective reagents we show that the two isoforms have distinct patterns of expression and distinct roles in cell proliferation, migration and tumor growth in vivo. In addition, based on metabolomics, functional and complementation assays, we propose a mechanistic basis for tumor cell dependency on glutaminase and the tumor adaptive response to glutaminase inhibition. Our data reveal the basis for the addiction of tumor subsets to glutaminase and elucidate the role of distinct isoforms of kidney-type glutaminase isoforms in cancer. Citation Format: Bonnie Liu, Kyung Song, Mandy Kwong, Min Gao, Rebecca Hong, Michelle Nannini, Deepak Sampath, Marcia Belvin, David Peterson, Marie Evangelista, Anneleen Daemen, Ron Firestein, Georgia Hatzivassiliou. Elucidating the role of distinct glutaminase isoforms in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4618. doi:10.1158/1538-7445.AM2013-4618

Abstract 1899: Metabolic vulnerability of breast cancer based on shifts in the expression of metabolic enzyme isoforms.

Abstract Background & Purpose: Isoforms of metabolic enzymes share similar catalytic activities. Most cells express several isoforms that likely represent redundancy and contribute robustness to biochemical processes. The distribution of isoforms varies by tissue and differentiation stage and may be altered during neoplastic transformation. Our hypothesis is iosenzyme expression changes in neoplastic cells, particularly the loss of isoenzyme diversity, can render cancer cells more vulnerable to metabolism targeted therapies. Our goal was to identify isoenzyme expression shifts in breast cancer compared to normal breast tissue to define metabolic processes in the cancer that rely on the expression of only one or a few enzyme isoforms due to loss of isoenzyme diversity. Methods: Metabolic enzymes (n=1,267) were identified in the KEGG database and we established pair-wise isoform expression distributions in breast cancer gene expression data sets (n=1,081 cancers) and in 40 normal breast samples. We looked for isoenzyme pairs that showed loss of expression in one isoform in cancer compared to normal while the other isoform was preserved or overexpressed. We assessed the isoform expression relationships in breast cancer cell lines (n=18) to identify experimental models for functional studies and used siRNAs to knock down the preserved isoform in cell lines that showed loss of the other isoform. Results: We identified 98 pairs of isoenzymes that showed reduced expression in one isoform in at least 20% of cancer patients compared to normal tissue (< 3 standard deviations below the mean of normal expression, the lower boundary of the same gene in normal tissues), while the expression of the second, larger than its lower boundary in normal tissues. These included PAPP2B/PAPP2C, COX7A1/COX7A2, ALDOC/ALDOA, LDHB/LDHA, and ME3/ME2 where the first member of the pair showed reduced expression in 83.7%, 48.2%, 33.0%, 49.4%, and 46.9% of cancers, respectively. Overall cancers shifted towards expressing isoforms that are normally high in muscle for many energy metabolism related enzymes. We tested the functional implication of knocking down ME2 (malic enzyme 2) expression in MDA435 cells that showed reduced ME3 expression but high ME2 levels. We observed a 54.0%, growth inhibition compared to scrambled siRNA control. In the control cell lines, MDA231 and MDA 468 that have preserved ME3 and ME2 expression, ME2 knockdown had no significant growth inhibitory effect. Conclusions: We demonstrate shifts in isoenzyme expression in breast cancers compare to normal breast. These shifts suggest adaptation to hypoxic environment, rapid metabolism and potential metabolic vulnerabilities. Our siRNA experiments with anti ME2 siRNA provide proof of concept that loss of isoenzyme diversity and primary reliance on a single or few isoenzymes to provide a metabolic function in neoplastic cells may be exploited therapeutically. Citation Format: Weiwei Shi, Aihua Gong, Tingting Jiang, Rene Natowicz, Lajos Pusztai. Metabolic vulnerability of breast cancer based on shifts in the expression of metabolic enzyme isoforms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1899. doi:10.1158/1538-7445.AM2013-1899

Keratin 8 and 18 Loss in Epithelial Cancer Cells Increases Collective Cell Migration and Cisplatin Sensitivity through Claudin1 Up-regulation

Keratins 8 and 18 (K8/18) are simple epithelial cell-specific intermediate filament proteins. Keratins are essential for tissue integrity and are involved in intracellular signaling pathways that regulate cell response to injuries, cell growth, and death. K8/18 expression is maintained during tumorigenesis; hence, they are used as a diagnostic marker in tumor pathology. In recent years, studies have provided evidence that keratins should be considered not only as markers but also as regulators of cancer cell signaling. The loss of K8/18 expression during epithelial-mesenchymal transition (EMT) is associated with metastasis and chemoresistance. In the present study, we investigated whether K8/18 expression plays an active role in EMT. We show that K8/18 stable knockdown using shRNA increased collective migration and invasiveness of epithelial cancer cells without modulating EMT markers. K8/18-depleted cells showed PI3K/Akt/NF-κB hyperactivation and increased MMP2 and MMP9 expression. K8/18 deletion also increased cisplatin-induced apoptosis. Increased Fas receptor membrane targeting suggests that apoptosis is enhanced via the extrinsic pathway. Interestingly, we identified the tight junction protein claudin1 as a regulator of these processes. This is the first indication that modulation of K8/18 expression can influence the phenotype of epithelial cancer cells at a transcriptional level and supports the hypothesis that keratins play an active role in cancer progression.

PL-13 Tissue factor isoforms in cancer and coagulation: May the best isoform win

Tissue factor (TF), the trigger of blood coagulation, is a 47 kDa membrane protein that also impacts on non-hemostatic processes, such as atherosclerosis, primary tumor growth and metastasis. TF binding to its ligand FVIIa induces activation of protease-activated receptor-2 and this event is thought to considerably influence atherosclerosis and tumor angiogenesis. TF-dependent activation of the coagulation cascade, rather than PAR-2 activation, then leads to the potentiation of metastasis. Importantly, a soluble alternatively spliced isoform of TF (asTF) has been discovered, but the function of asTF in hemostatic and non-hemostatic events is poorly understood. In this review, we aim to present a side-by-side evaluation of normally-spliced, full length TF (flTF) and asTF with regard to coagulant function, atherosclerosis, tumor progression and malignancy-associated thrombosis.

Regulation and function of the RSK family of protein kinases

The RSK (90 kDa ribosomal S6 kinase) family comprises a group of highly related serine/threonine kinases that regulate diverse cellular processes, including cell growth, proliferation, survival and motility. This family includes four vertebrate isoforms (RSK1, RSK2, RSK3 and RSK4), and single family member orthologues are also present in Drosophila and Caenorhabditis elegans. The RSK isoforms are downstream effectors of the Ras/ERK (extracellular-signal-regulated kinase) signalling pathway. Significant advances in the field of RSK signalling have occurred in the past few years, including several new functions ascribed to the RSK isoforms, the discovery of novel protein substrates and the implication of different RSK isoforms in cancer. Collectively, these new findings increase the diversity of biological functions regulated by RSK, and highlight potential new directions of research. In the present paper, we review the structure, expression and activation mechanisms of the RSK isoforms, and discuss their physiological roles on the basis of established substrates and recent discoveries.

VEGF gene alternative splicing: pro- and anti-angiogenic isoforms in cancer

Tumor growth and progression depend on angiogenesis, a process of new blood vessels formation from a preexisting vascular endothelium. Tumors promote angiogenesis by secreting or activating angiogenic factors that stimulate endothelial proliferation and migration and capillary morphogenesis. The newly formed blood vessels provide nutrients and oxygen to the tumor, increasing its growth. Thus, angiogenesis plays a key role in cancer progression and development of metastases. An important growth factor that promotes angiogenesis and participates in a variety of physiological and pathological processes is the vascular endothelial growth factor (VEGF-A or VEGF). Overexpression of VEGF results in increased angiogenesis in normal and pathological conditions. The existence of an alternative site of splicing at the 3' untranslated region of the mRNA results in the expression of isoforms with a C-terminal region which are downregulated in tumors and may have differential inhibitory effects. This suggests that control of splicing can be an important regulatory mechanism of angiogenesis in cancer.

Fine Tuning of Protein Kinase C (PKC) Isoforms in Cancer: Shortening the Distance from the Laboratory to the Bedside

The serine/threonine protein kinase C (PKC) family was first identified as intracellular receptor(s) for the tumor promoting agents phorbol esters. Thirty years after the discovery of PKC, the role of specific PKC isoforms has been described in relationship with an altered pattern of expression in different types of cancer and a good number of small molecule inhibitors (inhibitory peptides, antisense oligonucleotides or natural compounds) targeting PKC are now available. Despite all these achievements and a huge amount of basic research studies on the biochemical regulation of PKC, there has been a delay in clinical trials with drugs targeting PKC function. This delay is easily explained taking into account the extreme biological complexity of the PKC family of isoforms and the incomplete understanding of the specific role of each PKC isozyme in different types of cancers. Some of the difficulties in developing pharmacological compounds selectively tuning the different PKCs have started to be overcome. In this review, the growing evidences of the role of the PKC isoforms α, βII, δ, ε, ζ and ι is in promoting or counteracting tumor progression will be discussed in relationship with promising therapeutic perspectives.

Alternative splicing variants of carbonic anhydrase IX in human non-small cell lung cancer

In human cancers, carbonic anhydrase IX (CAIX) contributes to maintain intracellular and extracellular pH under hypoxic conditions, but also influences regulation of cell proliferation and tumor progression. CaIX was previously indicated as an independent prognostic marker in non-small cell lung carcinoma (NSCLC). Very recently a CAIX alternative splicing isoform, generating a transcript lacking of exons 8–9, was detected in cancer cells independently from the levels of hypoxia. This alternative splicing (AS) generates a truncated protein lacking the transmembrane region, the intracellular tail and the C-terminal of the catalytic domain and competes with the full-length (FL) isoform in the regulation of the extracellular pH, mainly in a mild hypoxic status. In the present study we measured the mRNA expression of FL and AS CAIX isoforms in 101 NSCLC and in paired not affected tissues. The two isoforms were coexpressed in all NSCLC and normal tissues but while AS mRNA was prevalent in normal tissues (66 ± 3%), the FL isoform was higher in NSCLC (58 ± 2%, p = 0.001). FL mRNA, but not AS, was statistically increased in NSCLC ( p = 0.01) and showed a statistical association with lymphnode involvement ( p = 0.009) and tumor stage ( p = 0.04). Global survival analysis of cancer/related death showed that high levels of FL mRNA were predictive of unfavorable outcome ( p < 0.0001) and shorter disease-free survival ( p < 0.0001). Multivariate analysis indicated that FL is an independent prognostic factor for overall survival and higher levels of mRNA in NSCLC sensibly increase hazard ratio (∼sixfold). In conclusion, our results seems to indicate that, at least in NSCLC, FL CAIX is the most accurate surrogate of hypoxic stress and represents the only variant with a prognostic role. These data indicate the importance of a separate measurement of the two isoforms in cancer and the need of an accurate re-evaluation of most studies on the clinical role of CAIX in cancer diagnosis.