Head and neck squamous cell carcinoma (HNSCC) develops in mucous membranes that line the mouth, throat and sinuses. It is the sixth most common cancer worldwide with more than 600,000 new cases diagnosed every year. The 5 year survival of HNSCC patients is less than 50% after initial diagnosis, while median survival after relapse or metastasis is less than one year. Unfortunately, the prognosis of HNSCC has not changed in the last two decades, mostly due to the poor understanding of the mechanism of this disease. New therapeutic targets and agents are in urgent need.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20662号 / 医博第4272号 / 新制||医||1024(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 松田 道行, 教授 原田 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Abstract Colorectal carcinoma (CRC) is an important cause of cancer-associated deaths. About 30–50% of CRCs show KRAS or BRAF mutation. In many cancers, anoikis, i.e. apoptosis induced by loss of extracellular matrix (ECM) contact, is disturbed. Anoikis resistance is essential for the formation of metastases, and since anoikis resistance assessment is based on in vitro cell cultures, the prognostic value of anoikis resistance is largely unknown. We aimed to identify the histopathological features indicating anoikis resistance in CRC and analyze their prognostic value. The roles of BRAF and KRAS mutations and survivin in anoikis resistance were analyzed and 3-D cell culture was used to model the histopathology of anoikis resistant (AR) structures. The two cohorts of CRC cases used in the study consisted of 62 (series 1) and 137 patients (series 2). Immunohistochemistry for ECM proteins enabled identification of tumor cells with and without ECM contact, and in both populations, apoptosis was determined with staining for caspase-cleaved keratin 18. Based on absence of ECM contact and decreased apoptosis rate, we identified micropapillary (MIP), cribriform and solid structures to represent the putative AR populations. High areal density of AR structures associated independently with short survival and was an independent prognostic factor. MIPs showed lower survivin expression, proliferation and apoptosis rates than non-MIP cells, and low apoptosis rate was associated with poor prognosis in stage I and II cases. For 3-D in vitro model of AR structures, we transfected Caco-2 cells with mutated KRAS or BRAF genes; both induced anoikis resistance as measured with Annexin V test in suspension culture. In 3-D cultures, native Caco-2 cells formed polarized cysts. In contrast, mutated cell lines formed partially filled cysts or solid structures, and inverted polarity in KRAS mutant cells. In conclusion, it is possible to identify putative AR structures by conventional histopathology and their number is associated with poor prognosis. MIPs represent a distinct subpopulation of CRC cells with features of quiescence. KRAS and BRAF mutations induce anoikis resistance in Caco-2 cells. In 3-D cultures, oncogenes KRAS and BRAF induce solid structures and cell piling, with structural resemblance to putative AR structures observed by histopathology. The mutated Caco-2 cells thus serve as a model to study the manifestation of anoikis resistance as a distinct histological feature with oncological significance. / Tiivistelmä Paksu- ja peräsuolisyöpä on yleinen syöpäkuoleman aiheuttaja. KRAS- tai BRAF-geenien mutaatio todetaan 30–50 prosentissa suolisyövistä. Anoikis tarkoittaa apoptoosia, jonka käynnistää solun irtoaminen soluväliaineesta. Anoikisresistenssi on etäpesäkkeen synnyn edellytys. Anoikisresistenssiä voidaan todeta vain soluviljelyssä, joten sen merkitystä syövässä in vivo ei ole aiemmin arvioitu. Tässä työssä pyrittiin tunnistamaan anoikisresistenssiin viittaavat muutokset histopatologisista suolisyöpänäytteistä ja selvittämään niiden vaikutusta potilaan ennusteeseen. Lisäksi tutkittiin BRAF- ja KRAS-mutaatioiden yhteyttä anoikisresistenssiin ja mallinnettiin anoikisresistenttejä (AR) rakenteita kolmiulotteisessa soluviljelmässä. Potilasaineisto koostui 199 suolisyöpäpotilaasta. Kudosleikkeistä värjättiin soluväliaineen komponentteja sekä määritettiin apoptoottiset ja jakautuvat solut (M30- ja Ki-67-värjäykset). AR-solupopulaatioiden tunnistamisessa käytettiin kriteereinä soluväliainekontaktin puuttumista ja vähentynyttä apoptoositiheyttä. AR-populaatioiksi osoittautuivat mikropapillaariset (MIP), seulamaiset ja solidit rakenteet. Näiden rakenteiden korkea kokonaisesiintyvyys osoittautui itsenäiseksi huonon ennusteen tekijäksi. MIP-rakenteissa surviviinin ilmentyminen ja apoptoosi- ja proliferaatiotiheys olivat vähentyneet muihin kasvainsoluihin verrattuna. Lisäksi apoptoottisten solujen pieni määrä MIP-rakenteissa liittyi huonoon ennusteeseen paikallisessa syövässä. Mallinnusta varten Caco-2 solut transfektoitiin mutatoiduilla KRAS- tai BRAF-geeneillä. Onkogeenien transfektion todettiin indusoivan anoikisresistenssiä. Kolmiulotteisessa soluviljelyssä polarisoituneet Caco-2 solut muodostivat säännöllisiä rauhasmaisia rakenteita. Onkogeeneillä transfektoidut solut muodostivat puolestaan osittain tai kokonaan täyttyneitä rakenteita ja KRAS-transfektio aiheutti solujen polariteetin kääntymistä. Havainnot osoittavat, että anoikisresistenssiä edustavat rakenteet voidaan tunnistaa kudosleikkeestä ja niiden runsas määrä viittaa huonoon ennusteeseen. MIP-rakenteissa todettiin lepotilan (quiescence) piirteitä. KRAS- ja BRAF-mutaatiot aiheuttavat Caco-2 soluissa anoikisresistenssiä. Kolmiulotteisissa soluviljelmissä onkogeenien vaikutus näkyy solujen pinoutumisena, mikä muistuttaa syöpäkudosnäytteissä todettuja AR-rakenteita. Tulosten perusteella modifioituja Caco-2 soluja voidaan hyödyntää anoikisresistenssin mallintamiseen ja tarkempien mekanismien tutkimiseen.

Cancer progression is a multi-step process requiring cells to acquire specific properties that aid the neoplastic growth. One such property is the ability to survive in the absence of matrix-attachment, a critical necessity for cells to traverse in circulation and seed metastases. Therefore, understanding the signalling mechanisms that protect cells from undergoing death in matrix-deprived condition, termed as anoikis, is important. We have used two systems to study this, one involving experimental transformation model, and another involving cancer cell lines. In the in vitro transformation model system involving the serial introduction of oncogenes, the ability to survive in anchorage-independent condition and generate spheres/colonies was dependent on the presence of the Simian Virus Small T antigen, SV40 ST. We identified that the viral antigen mediates its effects, at least in part, by activating the master metabolic regulator and cellular stress kinase AMP-activated protein kinase (AMPK) leading to maintenance of energy homeostasis. Consistent with this, our lab has previously identified both activation of AMPK upon matrix-deprivation in breast cells, as well as its requirement for survival under these conditions. However, a pathway often associated with survival under matrix-deprivation is the PI3K/Akt pathway. Surprisingly, we observed an AMPK-dependent decrease in Akt activity under conditions of matrix-detachment. Since this was contrary to the general notion, we probed deeper into a possible crosstalk between these two kinases. Our work revealed that AMPK activation in suspension inhibits Akt via upregulation of a known Akt phosphatase, pleckstrin homology domain leucinrich repeat protein phosphatise (PHLPP). We further show that the AMPK-PHLPP-Akt signalling axis is important for anoikis-resistance and metastasis. In addition, our results point to a yet unidentified protumorigenic role of PHLPP in breast cancer progression. With an aim to identify cellular proteins differentially regulated upon AMPK activation in breast cancer cells, we undertook a proteomics approach. Using 2-dimensional gel electrophoresis followed by mass spectrometric analysis, we identified some candidate proteins. We have validated the increase in levels of one of these proteins, annexin A2, in cancer cells upon AMPK activation. In summary, the present study unveils novel oncogenic functions of AMPK in cancer cells under the stress of matrix-deprivation. Furthermore, our results elucidate a double-negative feedback loop between two critical cellular kinases AMPK and Akt, and also identify a novel pro-tumorigenic role of PHLPP in breast cancer. In addition, we identify PHLPP and annexin A2 as novel proteins upregulated by AMPK in cancer cells. Thus, our results begin to identify pathways utilised by cancer cells to aid anchorage-independent growth, a critical step for cancer metastasis. Based on our results, inhibition of AMPK or perturbation of signalling axes involving AMPK, and PHLPP or annexin A2 might be considered as novel therapeutic approaches to combat cancer progression