Novel therapeutic targets in gastrointestinal stromal tumor

Background: Gastrointestinal stromal tumor (GIST) is one of the most common types of soft tissue sarcoma. The molecular mechanisms of GISTs are incompletely understood though the importance of KIT or platelet-derived growth factor α (PDGFRA) signaling in GIST is evident. The molecular mechanisms beyond KIT and PDGFRA signaling are incompletely understood. Tyrosine kinase inhibitors and especially imatinib, an inhibitor of KIT, PDGFRA and BCL-ABL, revolutionized the systemic treatment of GIST. However, advanced GISTs usually eventually progress on tyrosine kinase inhibitors, often because of secondary KIT mutations. There is a need for novel effective agents for the treatment of patients with GIST. Experimental design: The GIST gene expression profile was investigated in an in silico transcriptome database comprising of human tissue and cancer samples. Two GIST cell lines were screened for sensitivity to 217 anti-cancer compounds. SLUG, ITGA4, PDE3A and PDE3B expression was studied using immunohistochemistry on tissue microarrays (TMA). We used three different clinical cancer patient series: the first was a series that consisted of samples from 630 tumors from the archives of the Department of Pathology, Helsinki University Hospital; the second was a population-based cohort consisting of GIST patients who were treated with surgery in Western Sweden from 1983 through 2000; and the third series consisted of high risk GIST patients who were entered to the Scandinavian Sarcoma Group (SSG) XVIII/Arbeitsgemeinschaft Internistische Onkologie (AIO) adjuvant trial. The effects of SLUG, ITGA4 and PDE3 knockdown and selective ITGA4 and PDE3 inhibitors were investigated in three GIST cells lines. The efficacy of a PDE3 inhibitor, anagrelide, was investigated in patient-derived xenograft mouse models. Results: SLUG was expressed in 25.0 %, ITGA4 in 52.3 %, PDE3A in 90.9 % and PDE3B in 60.0 % of the GISTs investigated. Expression of these proteins were also detected in some other human tumor types, but usually much less frequently. SLUG and ITGA4 expression were associated several factors linked with unfavorable prognosis. SLUG expression was associated significantly also with unfavorable recurrence-free survival both when the patients were treated with surgery alone and when treated with surgery followed by adjuvant imatinib. ITGA4 expression was associated with unfavorable GIST-specific survival and overall survival in a patient population treated with surgery alone. PDE3A and PDE3B expression had no significant associations with the clinicopathological factors studied, RFS or overall survival. SLUG and PDE3 downregulation inhibited cell proliferation and induced apoptosis in GIST cell lines, whereas ITGA4 inhibition decreased GIST cell invasion. Anagrelide reduced or stabilized tumor growth in several GIST xenograft mouse models. Conclusions: SLUG, ITGA4 and PDE3s are frequently expressed in GISTs. They are likely important factors in the molecular pathogenesis of GIST, and may influence their clinical behavior. As ITGA4 and PDE3s can be targeted with specific inhibitors, they could potentially be therapeutic targets in GIST. SLUG may mediate pro-survival signaling in GIST. Some PDE3 inhibitors, such as anagrelide, warrant more study as potential therapeutic agents in GIST.Tutkimuksen tausta: Gastrointestinaalinen stroomakasvain (GIST) on yksi yleisimmistä pehmytkudossarkoomista. GIST-kasvainten molekulaariset mekanismit ovat vielä vaillinaisesti tunnettu, vaikka KIT- ja PDGFRA- tyrosiinikinaasien keskeinen rooli GIST-kasvaimissa onkin hyvin selvitetty. Tyrosiinikinaasi-inhibiittorit ja erityisesti imatinibi, joka estää KIT-, PDGFRA- ja BCL-ABL-tyrosiinikinaasien toimintaa, on mullistanut GIST-kasvainten hoidon. Tästä huolimatta pitkälle edennyt GIST-kasvain yleensä uusii, tyrosiinikinaasi-inhibittorihoidosta huolimatta, johtuen sekundäärisistä mutaatioista. Tästä syystä GIST-kasvainten hoitoon tarvitaan uusi tehokkaita lääkeaineita. Materiaalit ja menetelmät: GIST-kasvainten geenien ilmentymisprofiilit tutkittiin in silico transkriptomitietokannassa, joka koostui ihmisten normaalikudoksista sekä syöpäkudoksista. 217 syöpälääkkeen vaikutus testattiin kahdessa GIST-kasvainsolulinjassa. SLUG-, ITGA4-, PDE3A- ja PDE3B-proteiinien ilmentymistä tutkittiin immunohistokemiallisilla analyyseillä. Proteiinien ilmentymistä tutkittiin kolmessa eri syöpäpotilassarjassa. Ensimmäinen sarja koostui 630 syöpäkudosnäytteestä, jotka oli kerätty Helsingin yliopiston patologian laitoksen arkistoista. Toinen sarja koostui GIST-potilaista, jotka oli hoidettu leikkaushoidolla vuosien 1983 ja 2000 välillä Länsi-Ruotsissa. Kolmas sarja koostui korkean uusiutumisriskin omaavista GIST-potilaista, jotka osallistuivat Skandinavian sarkoomaryhmän (SSG) ja Arbeitsgemeinschaft Internistische Onkologien (AIO) liitännäishoitotutkimukseen. SLUG-, ITGA4- ja PDE3-proteiinien häirinnän sekä selektiivisten ITGA4- ja PDE3-proteiini-inhibiittorien vaikutusta tutkittiin kolmessa GIST-solulinjassa. PDE3-inhibiittori anagrelidin vaikutusta tutkittiin lisäksi hiiriin istutetuissa, ihmisistä peräisin olevissa syöpäkasvaimissa. Tulokset: SLUG-proteiini ilmentyi 25,0 %:ssa, ITGA4-proteiini 52,3 %:ssa, PDE3A-proteiini 90,9 %:ssa ja PDE3B-proteiini 60,0 %:ssa tutkituista GIST-kasvaimista. SLUG-proteiinin ja ITGA4-proteiinin ilmentyminen oli yhteydessä useisiin huonon ennusteen tekijöihin. SLUG-proteiinin ilmentyminen oli merkitsevästi yhteydessä myös kohonneeseen taudin uusiutumisriskiin, kun potilaita oli hoidettu ainoastaan kirurgisesti, mutta myös silloin, kun potilaita oli hoidettu kirurgian ja liitännäishoidon yhdistelmällä. ITGA4-proteiinin ilmentyminen oli yhteydessä epäsuotuisaan GIST-spesifiin elinaikaan ja kokonaiselinaikaan potilaissa, jotka oli hoidettu ainoastaan kirurgisesti. PDE3A- ja PDE3B-proteiinien ilmentyminen ei ollut merkitsevästi yhteydessä tutkittuihin kliinis-patologisiin tekijöihin, taudin uusiutumisriskiin tai potilaan kokonaiselinaikaan. SLUG- ja PDE3-proteiinien toiminnan häiritseminen esti solujen jakaantumista ja aiheutti solukuolemaa GIST-solulinjoissa, kun taas ITGA4-proteiinin esto vähensi GIST-solujen invaasiokykyä. PDE3-estäjä, anagrelide, pienensi kasvainten kokoa tai pysäytti kasvainten kasvun useissa GIST-hiirimalleissa. Johtopäätökset: SLUG-, ITGA4- ja PDE3-proteiinit ilmentyvät yleisesti GIST-kasvaimissa. Ne ovat luultavasti tärkeitä tekijöitä GIST-kasvainten synnyssä sekä toiminnassa ja vaikuttavat todennäköisesti niiden kliiniseen käyttäytymiseen. Koska ITGA4- ja PDE3-proteiineja vastaan löytyy spesifejä inhibiittoreita, ne voivat olla uusia mahdollisia lääkehoidon kohteita GIST-kasvaimissa. SLUG-proteiini saattaa ohjata signalointia, joka auttaa GIST-kasvaimia selviytymään. PDE3-inhibiittorit, kuten anagrelidi, vaativat lisätutkimuksia, jotta niiden todellinen kliininen potentiaali voidaan selvittää

Clinical relevance of integrin alpha 4 in gastrointestinal stromal tumours

The molecular mechanisms for the dissemination and metastasis of gastrointestinal stromal tumours (GIST) are incompletely understood. The purpose of the study was to investigate the clinical relevance of integrin alpha 4 (ITGA4) expression in GIST. GIST transcriptomes were first compared with transcriptomes of other types of cancer and histologically normal gastrointestinal tract tissue in the MediSapiens in silico database. ITGA4 was identified as an unusually highly expressed gene in GIST. Therefore, the effects of ITGA4 knock-down and selective integrin alpha 4 beta 1 (VLA-4) inhibitors on tumour cell proliferation and invasion were investigated in three GIST cell lines. In addition, the prognostic role of ITGA4 expression in cancer cells was investigated in a series of 147 GIST patients with immunohistochemistry. Inhibition of ITGA4-related signalling decreased GIST cell invasion in all investigated GIST cell lines. ITGA4 protein was expressed in 62 (42.2%) of the 147 GISTs examined, and expression was significantly associated with distant metastases during the course of the disease and several adverse prognostic features. Patients whose GIST expressed strongly ITGA4 had unfavourable GIST-specific survival and overall survival compared to patients with low or no ITGA4 expression. Taken together, ITGA4 is an important integrin in the molecular pathogenesis of GIST and may influence their clinical behaviour.Peer reviewe

Fibrinogen-like protein 2 in gastrointestinal stromal tumour

Gastrointestinal stromal tumour (GIST), the most common sarcoma of the gastrointestinal tract, can be treated effectively with tyrosine kinase inhibitors, such as imatinib. Cancer immune therapy has limited efficacy, and little is known about the immune suppressive factors in GISTs. Fibrinogen-like protein 2 (FGL2) is expressed either as a membrane-associated protein or as a secreted soluble protein that has immune suppressive functions. We found that GISTs expressed FGL2 mRNA highly compared to other types of cancer in a large human cancer transcriptome database. GIST expressed FGL2 frequently also when studied using immunohistochemistry in two large clinical series, where 333 (78%) out of the 425 GISTs were FGL2 positive. The interstitial cells of Cajal, from which GISTs may originate, expressed FGL2. FGL2 expression was associated with small GIST size, low mitotic counts and low tumour-infiltrating lymphocyte (TIL) counts. Patients whose GIST expressed FGL2 had better recurrence-free survival than patients whose GIST lacked expression. Imatinib upregulated FGL2 in GIST cell lines, and the patients with FGL2-negative GIST appeared to benefit most from long duration of adjuvant imatinib. We conclude that GISTs express FGL2 frequently and that FGL2 expression is associated with low TIL counts and favourable survival outcomes.Peer reviewe

SLUG transcription factor : a pro-survival and prognostic factor in gastrointestinal stromal tumour

Background: The SLUG transcription factor has been linked with the KIT signalling pathway that is important for gastrointestinal stromal tumour (GIST) tumourigenesis. Its clinical significance in GIST is unknown. Methods: Influence of SLUG expression on cell proliferation and viability were investigated in GIST48 and GIST882 cell lines. The association between tumour SLUG expression in immunohistochemistry and recurrence-free survival (RFS) was studied in two clinical GIST series, one with 187 patients treated with surgery alone, and another one with 313 patients treated with surgery and adjuvant imatinib. Results: SLUG downregulation inhibited cell proliferation, induced cell death in both cell lines, and sensitised GIST882 cells to lower imatinib concentrations. SLUG was expressed in 125 (25.0%) of the 500 clinical GISTs evaluated, and expression was associated with several factors linked with unfavourable prognosis. SLUG expression was associated with unfavourable RFS both when patients were treated with surgery alone (HR = 3.40, 95% CI = 1.67-6.89, P = 0.001) and when treated with surgery plus adjuvant imatinib (HR = 1.83, 95% CI = 1.29-2.60, P = 0.001). Conclusions: GIST patients with high tumour SLUG expression have unfavourable RFS. SLUG may mediate pro-survival signalling in GISTs.Peer reviewe

Prognostic impact of kallikrein-related peptidase transcript levels in prostate cancer

Peer reviewe

Anagrelide for Gastrointestinal Stromal Tumor

PURPOSE: Gastrointestinal stromal tumor (GIST) is a common type of soft-tissue sarcoma. Imatinib, an inhibitor of KIT, platelet-derived growth factor receptor alpha (PDGFRA), and a few other tyrosine kinases, is highly effective for GIST, but advanced GISTs frequently progress on imatinib and other approved tyrosine kinase inhibitors. We investigated phosphodiesterase 3 (PDE3) as a potential therapeutic target in GIST cell lines and xenograft models. EXPERIMENTAL DESIGN: The GIST gene expression profile was interrogated in the MediSapiens IST Online transcriptome database comprising human tissue and cancer samples, and PDE3A and PDE3B expression was studied using IHC on tissue microarrays (TMA) consisting of 630 formalin-fixed human tissue samples. GIST cell lines were screened for sensitivity to 217 anticancer compounds, and the efficacy of PDE inhibitors on GIST was further studied in GIST cell lines and patient-derived mouse xenograft models. RESULTS: GISTs expressed PDE3A and PDE3B frequently compared with other human normal or cancerous tissues both in the in silico database and the TMAs. Anagrelide was identified as the most potent of the PDE3 modulators evaluated. It reduced cell viability, promoted cell death, and influenced cell signaling in GIST cell lines. Anagrelide inhibited tumor growth in GIST xenograft mouse models. Anagrelide was also effective in a GIST xenograft mouse model with KIT exon 9 mutation that may pose a therapeutic challenge, as these GISTs require a high daily dose of imatinib. CONCLUSIONS: PDE3A and PDE3B are frequently expressed in GIST. Anagrelide had anticancer efficacy in GIST xenograft models and warrants further testing in clinical trials.status: publishe