Isoforms In Cancer Research Articles

Dysregulation of adenosine kinase isoforms in breast cancer.

Dysregulated adenosine signaling pathway has been evidenced in the pathogenesis of breast cancer. However, the role of adenosine kinase (ADK) in tumorigenesis remains unclear while it crucially regulates the removal and availability of adenosine. ADK has two isoforms that localize to discrete subcellular spaces: i.e., nuclear, long-isoform (ADK-L) and cytosolic, short-isoform (ADK-S). We hypothesized that these two ADK isoforms would be differentially expressed in breast cancer and may contribute to divergent cellular actions in cancer. In this study, we examined the expression profiles of ADK isoforms in breast cancer tissues from 46 patient and followed up with an in vitro investigation by knocking down the expression of ADK-L or ADK-S using CRISPR gene editing to evaluate the role of ADK isoform in cancer progression and metastasis of cultured triple-negative breast cancer cell line MDA-MB-231. We demonstrated that (i) ADK-L expression level was significantly increased in breast cancer tissues versus paired normal tissues adjacent to tumor, whereas the ADK-S expression levels were not significantly different between cancerous and normal tissues; (ii) CRISPR/Cas9-mediated downregulation of ADK isoforms, led to suppressed cellular proliferation, division, and migration of cultured breast cancer cells; (iii) ADK-L knockdown significantly upregulated gene expression of matrix metalloproteinase (ADAM23, 9.93-fold; MMP9, 24.58-fold) and downregulated expression of cyclin D2 (CCND2, -30.76-fold), adhesive glycoprotein THBS1 (-8.28-fold), and cystatin E/M (CST6, -16.32-fold). Our findings suggest a potential role of ADK-L in mitogenesis, tumorigenesis, and tumor-associated tissue remodeling and invasion; and the manipulation of ADK-L holds promise as a therapeutic strategy for aggressive breast cancer.

The Diverse Functions of Mutant 53, Its Family Members and Isoforms in Cancer.

The p53 family of proteins has grown substantially over the last 40 years. It started with p53, then p63, p73, isoforms and mutants of these proteins. The function of p53 as a tumour suppressor has been thoroughly investigated, but the functions of all isoforms and mutants and the interplay between them are still poorly understood. Mutant p53 proteins lose p53 function, display dominant-negative (DN) activity and display gain-of-function (GOF) to varying degrees. GOF was originally attributed to mutant p53′s inhibitory function over the p53 family members p63 and p73. It has become apparent that this is not the only way in which mutant p53 operates as a large number of transcription factors that are not related to p53 are activated on mutant p53 binding. This raises the question to what extent mutant p53 binding to p63 and p73 plays a role in mutant p53 GOF. In this review, we discuss the literature around the interaction between mutant p53 and family members, including other binding partners, the functional consequences and potential therapeutics.

HOX transcript antisense RNA (HOTAIR) in cancer.

Long noncoding RNAs (lncRNAs) have emerged as a new family of master regulators of cancer. The lncRNA HOX transcript antisense RNA (HOTAIR) is a prime example of an oncogenic trans-acting lncRNA. The expression of HOTAIR is elevated in a broad spectrum of cancers and is associated with metastasis and poor prognosis. HOTAIR governs fundamental biochemical and cellular processes via interactions with a variety of partners to promote proliferation, invasion, survival, drug resistance, and metastasis in preclinical studies of cancer. Here, we review the diagnostic and therapeutic potential of HOTAIR as well as the molecular mechanisms underlying the dysregulation of its expression and function in cancer. We also discuss the challenges to capitalizing on HOTAIR for more effective patient care along with future directions founded on the recent exciting advances in our knowledge of HOTAIR in cancer.

Netrin Family: Role for Protein Isoforms in Cancer

Netrins form a family of secreted and membrane-associated proteins. Netrins are involved in processes for axonal guidance, morphogenesis, and angiogenesis by regulating cell migration and survival. These processes are of special interest in tumor biology. From the netrin genes various isoforms are translated and regulated by alternative splicing. We review here the diversity of isoforms of the netrin family members and their known and potential roles in cancer.

Numb Isoforms Deregulation in Medulloblastoma and Role of p66 Isoform in Cancer and Neural Stem Cells.

Numb is an intracellular protein with multiple functions. The two prevalent isoforms, Numb p66 and Numb p72, are regulators of differentiation and proliferation in neuronal development. Additionally, Numb functions as cell fate determinant of stem cells and cancer stem cells and its abnormal expression has been described in several types of cancer. Involvement of deregulated Numb expression has been described in the malignant childhood brain tumor medulloblastoma, while Numb isoforms in these tumors and in cancer stem-like cells derived from them, have not been studied to date. Here we show that medulloblastoma stem-like cells and cerebellar neuronal stem cells (NSCs) express Numb p66 where its expression tampers stemness features. Furthermore, medulloblastoma samples evaluated in this study express decreased levels of Numb p66 while overexpressed Numb p72 compared with normal tissues. Our results uncover different roles for the two major Numb isoforms examined in medulloblastoma and a critical role for Numb p66 in regulating stem-like cells and NSCs maintenance.

The lipid products of phosphoinositide 3-kinase isoforms in cancer and thrombosis.

Our knowledge on the role of the different lipid messengers produced by phosphoinositide 3-kinases (PI3Ks) in normal and cancer cells as well as in platelets during arterial thrombosis has greatly expanded these last 15years. PI3Ks are a family of lipid kinases that catalyze the phosphorylation of the D3 position of the inositol ring of phosphoinositides to produce phosphatidylinositol 3-phosphate (PtdIns3P), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), and phosphatidylinositol-3,4,5 trisphosphate (PtdIns(3,4,5)P3). These D3-phosphoinositides act as intracellular messengers recruiting effector proteins involved in the control of diverse cellular functions including survival, proliferation, migration, membrane trafficking, and cytoskeleton dynamics. The current idea is that the different isoforms of PI3Ks produce specific pools of lipids that regulate in time and space, at the membrane/cytosol interface, the formation of appropriate functional protein complexes. Dysregulation of PI3K-dependent pathways is directly involved in the etiology of several pathologies including cancers where the PI3K/AKT/mTORC1 axis is frequently aberrantly activated. Moreover, PtdIns(3,4,5)P3 production has been shown to play an essential role in platelet functions, particularly in the formation of a stable platelet thrombus at high shear rate. Therefore, PI3Ks are attractive therapeutic targets in the treatment of cancer and arterial thrombosis. In this review, we will discuss the role of the different lipid products of PI3K isoforms in the context of cancer and thrombosis and the development of selective PI3Ks inhibitors in the treatment of these diseases.

Structure, Function, Involvement in Diseases and Targeting of 14-3-3 Proteins: An Update.

14-3-3 is a class of proteins able to interact with a multitude of targets by establishing protein-protein interactions (PPIs). They are usually found in all eukaryotes with a conserved secondary structure and high sequence homology among species. 14-3-3 proteins are involved in many physiological and pathological cellular processes either by triggering or interfering with the activity of specific protein partners. In the last years, the scientific community has collected many evidences on the role played by seven human 14-3-3 isoforms in cancer or neurodegenerative diseases. Indeed, these proteins regulate the molecular mechanisms associated to these diseases by interacting with (i) oncogenic and (ii) pro-apoptotic proteins and (iii) with proteins involved in Parkinson and Alzheimer diseases. The discovery of small molecule modulators of 14-3-3 PPIs could facilitate complete understanding of the physiological role of these proteins, and might offer valuable therapeutic approaches for these critical pathological states.

Abstract 2442: Simplified sequencing of full-length isoforms in cancer on the PacBio Sequel System

Abstract Tremendous flexibility is maintained in the human proteome via alternative splicing, and cancer genomes often subvert this flexibility to promote survival. Identification and annotation of cancer-specific mRNA isoforms is critical to understanding how mutations in the genome affect the biology of cancer cells. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. In cancer proteomics studies, the identification of biomarkers from mass spectroscopy data is often limited by incomplete gene isoform expression information to support protein to transcript mapping. The Iso-Seq™ protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences needed to discover biomarkers for early detection and cancer stratification, to fully characterize gene fusion events, and to elucidate drug resistance mechanisms. Knowledge of the complete isoform repertoire is also key for accurate quantification of isoform abundance. As most transcripts range from 1 - 10 kb, fully intact RNA molecules can be sequenced using SMRT® Sequencing without requiring fragmentation or post-sequencing assembly. However, some cancer research applications have presented a challenge for the Iso-Seq protocol, due to the combination of limited sample input and the need to deeply sequence heterogenous samples. Here we report the optimization of the Iso-Seq library preparation protocol for the PacBio Sequel platform and its application to cancer cell lines and tumor samples. We demonstrate how loading enhancements on the higher-throughput Sequel instrument have decreased the need for size fractionation steps, reducing sample input requirements while simultaneously simplifying the sample preparation workflow and increasing the number of full-length transcripts per SMRT Cell. The results highlight the potential for broader application of the Iso-Seq method to more comprehensively characterize alternative splicing in cancer. Citation Format: Meredith Ashby, Ting Hon, Elizabeth Tseng, Aparna Vedula, Tyson A. Clark. Simplified sequencing of full-length isoforms in cancer on the PacBio Sequel System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2442. doi:10.1158/1538-7445.AM2017-2442

Abstract 2367: Identification of ALK alternative transcription initiation in BRAF-negative metastatic melanoma patients

Abstract Background: Genomic alterations in the ALK tyrosine kinase, such as copy number gains, point mutations, and ALK gene fusions, have been described in a broad spectrum of malignancies. Recently a novel mechanism of ALK activation in melanoma was discovered in which ALK transcription was initiated from a de novo alternative transcription initiation (ATI) site in ALK intron 19 (ALK-ATI) [1]. In this study, we used whole transcriptome sequencing (WTS) to interrogate a cohort of clinical melanoma specimens in order to identify those that express ALK-ATI. Method: RNA-seq libraries of 33 BRAF-negative clinical metastatic melanoma specimens were prepared using the KAPA Stranded RNA-Seq Kit with RiboErase with 200ng RNA as input and sequenced on Illumina HiSeq 4000 at 2x75bp. The specimens were sequenced to an average of 69.4m total read pairs with 11.3m on-target distinct read pairs. Sequencing reads were mapped to the human genome and transcriptome, gene- and exon-level expressions were quantified. An algorithm to identify the ALK-ATI was developed based on assessing 1) transcription of intron 19 putative ATI region, 2) ALK relative RNA expression and 3) ALK 5’ to 3’ differential expression. In addition, immunohistochemistry (IHC) staining for ALK (using D5F3 antibody) was done on 10 of the 33 samples with adequate tumor material. Results: High coverage (median 30x) of intron 19 ATI region and high ALK relative expression (log ratio > 2) were observed in 4 of the 33 specimens tested, in which the ATI site is consistent with what had been reported in [1]. Among the 4 potential ALK-ATI positive cases, 3 have shown significant 3’ to 5’ overexpression of ALK. IHC staining were performed on the 4 putative ALK-ATI positive cases and 6 putative negative cases, and has confirmed ALK-ATI calls detected by WTS assay. Two of the ALK IHC positive cases harbor the highest ALK expression, and the other two ALK-ATI likely cases are ALK IHC weak positive. ALK-ATI occurred in cutaneous, mucosal, and unknown primary melanoma, and co-occurred with other driver mutations (NRAS, NF1, KIT). None of the above specimens had ALK rearrangements detected from DNAseq (by FoundationOne) or WTS. Conclusions: Our findings confirmed previously reported ALK activation through ALK-ATI in an independent melanoma cohort, and provided direct evidence that such events can be detected using whole transcriptome sequencing and align with IHC results in clinical FFPE tumor specimens. This confirmation together with future functional validation and clinical evidence may provide new therapeutic opportunities for BRAF-negative metastatic melanoma patients. 1. Wiesner, T. et al. Alternative transcription initiation leads to expression of a novel ALK isoform in cancer. Nature 526, 453-457 (2015). Citation Format: Shan Zhong, Christine Lovly, Christine Malboeuf, Kristina Brennan, Douglas Johnson, Kelsie Riemenschneider, Catherine Meador, Emily White, Geoff A. Otto, Doron Lipson, Philip Stephens, Vincent Miller, Jeffrey Ross, Jie He. Identification of ALK alternative transcription initiation in BRAF-negative metastatic melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2367. doi:10.1158/1538-7445.AM2017-2367

Protein kinase C (PKC) isoforms in cancer, tumor promotion and tumor suppression.

The AGC family of serine/threonine kinases (PKA, PKG, PKC) includes more than 60 members that are critical regulators of numerous cellular functions, including cell cycle and differentiation, morphogenesis, and cell survival and death. Mutation and/or dysregulation of AGC kinases can lead to malignant cell transformation and contribute to the pathogenesis of many human diseases. Members of one subgroup of AGC kinases, the protein kinase C (PKC), have been singled out as critical players in carcinogenesis, following their identification as the intracellular receptors of phorbol esters, which exhibit tumor-promoting activities. This observation attracted the attention of researchers worldwide and led to intense investigations on the role of PKC in cell transformation and the potential use of PKC as therapeutic drug targets in cancer diseases. Studies demonstrated that many cancers had altered expression and/or mutation of specific PKC genes. However, the causal relationships between the changes in PKC gene expression and/or mutation and the direct cause of cancer remain elusive. Independent studies in normal cells demonstrated that activation of PKC is essential for the induction of cell activation and proliferation, differentiation, motility, and survival. Based on these observations and the general assumption that PKC isoforms play a positive role in cell transformation and/or cancer progression, many PKC inhibitors have entered clinical trials but the numerous attempts to target PKC in cancer has so far yielded only very limited success. More recent studies demonstrated that PKC function as tumor suppressors, and suggested that future clinical efforts should focus on restoring, rather than inhibiting, PKC activity. The present manuscript provides some historical perspectives on the tumor promoting function of PKC, reviewing some of the observations linking PKC to cancer progression, and discusses the role of PKC in the pathogenesis of cancer diseases and its potential usage as a therapeutic target.

Phosphorylation Weakens but Does Not Inhibit Membrane Binding and Clustering of K-Ras4B.

K-Ras4B is one of the most frequently mutated Ras isoforms in cancer. The signaling activity of K-Ras4B depends on its localization to the plasma membrane (PM), which is mainly mediated by its polybasic farnesylated C-terminus. On top of the constitutive cycles that maintain the PM enrichment of K-Ras4B, conditional phosphorylation at Ser181 located within this motif has been found to be involved in regulating K-Ras4B's cell distribution and signaling activity. However, discordant observations have undermined our understanding of the role this phosphorylation plays. Here, we report an efficient strategy for producing K-Ras4B simultaneously bearing phosphate, farnesyl, and methyl modifications on a preparative scale, a very useful in vitro system when used in concert with model biomembranes. By using this system, we determined that phosphorylation at Ser181 does not fully inhibit membrane binding and clustering of K-Ras4B but reduces its membrane binding affinity, depending on membrane fluidity. In addition, phosphorylated K-Ras4B maintains tight association with its cytosolic shuttle protein PDEδ. After delivering K-Ras4B containing nonhydrolyzable phosphoserine mimetic into cells, the protein displayed a decreasing PM distribution compared with nonphosphorylable K-Ras4B, implying that phosphorylation might facilitate the dissociation of K-Ras4B from the PM. In addition, phosphorylation does not alter the localization of K-Ras4B in the liquid-disordered lipid subdomains of the membrane but slightly alters the thermotropic properties of K-Ras4B-incorporated membranes probably due to minor differences in membrane partitioning and dynamics. These results provide novel mechanistic insights into the role that phosphorylation at Ser181 plays in regulating K-Ras4B's distribution and activity.

Defining the role of the RSK isoforms in cancer.

The 90kDa ribosomal S6 kinase (RSK) family is a group of Ser/Thr protein kinases (RSK1-4) that function downstream of the Ras/mitogen-activated protein kinase (MAPK) signalling pathway. RSK regulates many substrates involved in cell survival, growth, and proliferation, and as such, deregulated RSK activity has been associated with multiple cancer types. RSK expression and activity are dysregulated in several malignancies, including breast, prostate, and lung cancer, and available evidence suggests that RSK may be a promising cancer therapeutic target. Current limitations include the lack of RSK inhibitors with suitable pharmacokinetics and selectivity toward particular isoforms. This review briefly describes the current knowledge on RSK activation and function, with a particular emphasis on RSK-dependent mechanisms associated with tumorigenesis and pharmacological inhibition.

Abstract 4772: Addiction to chaperones: Investigating the role of HSP70 isoforms in cancer

Abstract The heat shock protein 70kDa (HSP70) family of molecular chaperones are critical for the survival of cancer cells due to their antiapoptotic activity and role in protein homeostasis. Our laboratory has previously shown that silencing two members of this family (HSP72 and HSC70) causes tumour cell specific apoptosis and also sensitises cancer cells to HSP90 inhibitors. HSP70 has therefore become an appealing therapeutic target. The human HSP70 family comprises 17 genes and the individual roles of the isoforms in cancer are poorly understood. Here we use a systematic approach to determine the importance of HSP70 isoforms, and a selection of HSP70 binding partners, for the growth of cancer cells under stress. It was hypothesised that cancer cells are more dependent on HSP70 proteins when under stressed conditions, as encountered in the tumour environment. We used RNA interference (RNAi) to determine the effect of silencing HSP70 isoforms and binding partners on cancer cell viability, as measured by CellTiter-Blue®. The siRNA screens were carried out in human colon (RKO) and breast (BT474) cancer cells under serum deprivation, oxidative stress and hypoxia. The silencing of several HSP70 genes, including the HSP70 isoform HSPA5 (also known as BiP/Grp78), were found to cause a reduction in cancer cell viability under stress. Detailed validation of hits has been carried out to confirm their importance for cancer cell survival and validated hits may provide potentially important therapeutic targets for cancer drug discovery. Citation Format: Lorna AB Suckling, Marissa Powers, Swee Sharp, Paul Workman. Addiction to chaperones: Investigating the role of HSP70 isoforms in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4772.

Matrix metalloproteinase protein inhibitors: highlighting a new beginning for metalloproteinases in medicine

Matrix metalloproteinase protein inhibitors: highlighting a new beginning for metalloproteinases in medicine Vishnu Mohan,1 Dalit Talmi-Frank,1 Valeria Arkadash,2 Niv Papo,2 Irit Sagi1 1Department of Biological Regulation, Weizmann Institute of Science, Rehovot, 2Department of Biotechnology Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel Abstract: The development of therapeutic matrix metalloproteinase (MMP) inhibitors has evolved from broad-spectrum peptidomimetic inhibitors with deleterious side effects, to highly selective agents. These range from small molecules to antibodies, antisense inhibitors, and engineered N-terminal tissue inhibitors of metalloproteinase domain. The advances in inhibitor design along with promising new global molecular insights into MMP structures, the protease web, and the role of extracellular matrix in diseases have contributed toward a renewed interest in using MMPs as valid drug targets. This review aims to address the advances and challenges concerning the design, development, and current status of anti-MMP agents in this new era of post-broad-spectrum MMP inhibitors. Highly selective inhibitors of MMPs promise to usher in an era of specific targeting of diseased tissue proteolysis networks, with markedly reduced negative repercussions, and to uncover the molecular and mechanistic roles of MMP isoforms in cancer, inflammation, and infection. Keywords: matrix metalloproteinase inhibitors, MMPI, small molecules, MMP-inhibiting monoclonal antibodies, engineered N-TIMP, miRNA

Alternative Polyadenylation: Another Foe in Cancer.

Advancements in sequencing and transcriptome analysis methods have led to seminal discoveries that have begun to unravel the complexity of cancer. These studies are paving the way toward the development of improved diagnostics, prognostic predictions, and targeted treatment options. However, it is clear that pieces of the cancer puzzle are still missing. In an effort to have a more comprehensive understanding of the development and progression of cancer, we have come to appreciate the value of the noncoding regions of our genomes, partly due to the discovery of miRNAs and their significance in gene regulation. Interestingly, the miRNA-mRNA interactions are not solely dependent on variations in miRNA levels. Instead, the majority of genes harbor multiple polyadenylation signals on their 3' UTRs (untranslated regions) that can be differentially selected on the basis of the physiologic state of cells, resulting in alternative 3' UTR isoforms. Deregulation of alternative polyadenylation (APA) has increasing interest in cancer research, because APA generates mRNA 3' UTR isoforms with potentially different stabilities, subcellular localizations, translation efficiencies, and functions. This review focuses on the link between APA and cancer and discusses the mechanisms as well as the tools available for investigating APA events in cancer. Overall, detection of deregulated APA-generated isoforms in cancer may implicate some proto-oncogene activation cases of unknown causes and may help the discovery of novel cases; thus, contributing to a better understanding of molecular mechanisms of cancer. Mol Cancer Res; 14(6); 507-17. ©2016 AACR.

A New View of Ras Isoforms in Cancers.

Does small GTPase K-Ras4A have a single state or two states, one resembling K-Ras4B and the other N-Ras? A recent study of K-Ras4A made the remarkable observation that even in the absence of the palmitoyl, K-Ras4A can be active at the plasma membrane. Importantly, this suggests that K-Ras4A may exist in two distinct signaling states. In state 1, K-Ras4A is only farnesylated, like K-Ras4B; in state 2, farnesylated and palmitoylated, like N-Ras. The K-Ras4A hypervariable region sequence is positively charged, in between K-Ras4B and N-Ras. Taken together, this raises the possibility that the farnesylated but nonpalmitoylated state 1, like K-Ras4B, binds calmodulin and is associated with colorectal and other adenocarcinomas like lung cancer and pancreatic ductal adenocarcinoma. On the other hand, state 2 may be associated with melanoma and other cancers where N-Ras is a major contributor, such as acute myeloid leukemia. Importantly, H-Ras has two, singly and doubly, palmitoylated states that may also serve distinct functional roles. The multiple signaling states of palmitoylated Ras isoforms question the completeness of small GTPase Ras isoform statistics in different cancer types and call for reevaluation of concepts and protocols. They may also call for reconsideration of oncogenic Ras therapeutics.

The role of wild type RAS isoforms in cancer

The role of wild type RAS isoforms in cancer

Targeted Proteomics Enables Simultaneous Quantification of Folate Receptor Isoforms and Potential Isoform-based Diagnosis in Breast Cancer.

The distinct roles of protein isoforms in cancer are becoming increasingly evident. FRα and FRβ, two major isoforms of the folate receptor family, generally have different cellular distribution and tissue specificity. However, the presence of FRβ in breast tumors, where FRα is normally expressed, complicates this situation. Prior to applying any FR isoform-based diagnosis and therapeutics, it is essential to monitor the expression profile of FR isoforms in a more accurate manner. An LC-MS/MS-based targeted proteomics assay was developed and validated in this study because of the lack of suitable methodology for the simultaneous and specific measurement of highly homologous isoforms occurring at low concentrations. FRα and FRβ monitoring was achieved by measuring their surrogate isoform-specific peptides. Five human breast cell lines, isolated macrophages and 60 matched pairs of breast tissue samples were subjected to the analysis. The results indicated that FRβ was overexpressed in tumor-associated macrophages (TAMs) but not epithelial cells, in addition to an enhanced level of FRα in breast cancer cells and tissue samples. Moreover, the levels of the FR isoforms were evaluated according to the histology, histopathological features and molecular subtypes of breast cancer. Several positive associations with PR/ER and HER2 status and metastasis were revealed.

Alternative transcription initiation leads to expression of a novel ALK isoform in cancer

Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ∼11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALK(ATI). In ALK(ATI)-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites. ALK(ATI) is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALK(ATI) transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALK(ATI) stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALK(ATI), suggesting that patients with ALK(ATI)-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.

TGF-ß isoforms in cancer: Immunohistochemical expression and Smad-pathway-activity-analysis in thirteen major tumor types with a critical appraisal of antibody specificity and immunohistochemistry assay validity.

The literature on TGF-ß in cancer including data on the expression or activation of TGF-ß pathway components in specific tumors types is steadily growing. However, no systematic and uniform analysis exists reporting expression levels of the main TGF-ß pathway components across the most frequent tumor types. We used a standardized immunohistochemical assay investigating TGF-ß isoform expression and pathway activation across 13 different tumor types and corresponding non-neoplastic tissues. The study was performed on tissue microarrays allowing for the parallel analysis of a total of 1638 human tumor samples. TGF-ß1, TGF-ß2 and p-Smad2/3 were substantially expressed in multiple cancers widening the options for TGF-ß isoform directed therapies. Of note, TGF-ß antigens appear to be expressed in an individual manner pointing towards a need for patient preselection for TGF-β isoform specific treatment. Yet, a thorough investigation of antibody specificity and assay validity revealed that immunohistochemistry did not correlate with other detection methods on mRNA or protein level in all instances. As such, with the currently available means (i.e. antibodies tested) a stratification of patients within clinical trials for TGF-ß directed antisense therapies based upon TGF-β immunohistochemistry alone has to be interpreted with caution and should be carefully evaluated in combination with other parameters.