Factors Affecting Cancer Behavior with Special Reference to Lymphatic Vessels, Macrophages, EGFR, and Pim-1 in Colorectal Cancer

Syövän käyttäytymiseen ja ennusteeseen vaikuttavat monet tekijät, muun muassa muutokset syöpäsoluissa sekä kasvainta ympäröivässä mikroympäristössä. Tutkimuksen tavoitteena oli tutkia uusia prediktiivisiä ja prognostisia ennustetekijöitä syöpäsoluissa (EGFR geenikopiomäärä, EGFR, onkogeeni pim-1) sekä syöpäkasvaimen mikroympäristöön kuuluvissa imuteissä (CLEVER-1, podoplaniini), makrofageissa (CD68, CLEVER-1) ja T lymfosyyteissä (CD3) kolorektaalisyövässä. Lisäksi tutkittiin imuteiden molekulaarisia ominaisuuksia tarkemmin (CD73, LYVE-1, podoplaniini) kuten myös lymfosyyttien ja dendriittisolujen liikennöintiä imuteissä. Tutkimustulokset osoittavat että korkea Pim-1 ekspressiotaso, suuri peritumoraalinen CD68+ makrofagimäärä sekä varhaisen vaiheen taudissa suuri CLEVER-1+ peritumoraalinen makrofagimäärä ovat hyvän ennusteen tekijöitä kolorektaalisyövässä. Metastaattisessa taudissa sen sijaan suuri määrä CLEVER-1+ makrofageja, sekä intra- että peritumoraalisesti, liittyy huonoon tautiennusteeseen. EGFR geenikopiomäärä, EGFR proteiinipitoisuuden ohjaaman hopea in situ hybridisaatiomenetelmän avulla määritettynä, ennusti vastetta anti-EGFR hoidolle metastaattisessa kolorektaalisyövässä tarkemmin kuin nykyisin rutiinisti käytössä oleva KRAS määritys. Lisäksi havaittiin että imutiet ovat monimuotoisia imutiemarkkeri ekspressionsa suhteen sekä normaali- että syöpäkudoksissa. CD73 molekyylin funktio imuteissä poikkesi selvästi molekyylin funktiosta verisuonissa. Yhteenvetona voidaan todeta että kolorektaalisyövän ennusteeseen vaikuttavien tekijöiden merkitys vaihtelee taudin levinneisyysasteen sekä imuteiden että makrofagien sijainnin perusteella. Korkea Pim-1 ilmentyminen on yhteydessä hyvään kolorektaalisyöpäennusteeseen. Lisäksi EGFR geenikopiomäärä osoittautui lupaavaksi uudeksi prediktiiviseksi ennustetekijäksi KRAS villintyypin metastaattista kolorektaalisyöpää sairastavilla potilailla.

The prognostic and predictive value of selected biomarkers in colorectal cancer

Tissue-based biomarkers are studied to receive information about the pathologic processes and cancer outcome, and to enable development of patient-tailored treatments. The aim of this study was to investigate the potential prognostic and/or predictive value of selected biomarkers in colorectal cancer (CRC). Group IIA secretory phospholipase A2 (IIA PLA2) expression was assessed in 114 samples presenting different phases of human colorectal carcinogenesis. Securin, Ki-67, CD44 variant 6 (CD44v6), aldehyde dehydrogenase 1 (ALDH1) and β-catenin were studied in a material including 227 rectal carcinoma patients treated with short-course preoperative radiotherapy (RT), long-course preoperative (chemo)RT (CRT) or surgery only. Epidermal growth factor receptor (EGFR) gene copy number (GCN), its heterogeneity in CRC tissue, and association with response to EGFR-targeted antibodies cetuximab and panitumumab were analyzed in a cohort of 76 metastatic CRC. IIA PLA2 expression was decreased in invasive carcinomas compared to adenomas, but did not relate to patient survival. High securin expression after long-course (C)RT and high ALDH1 expression in node-negative rectal cancer were independent adverse prognostic factors, ALDH1 specifically in patients treated with adjuvant chemotherapy. The lack of membranous CD44v6 in the rectal cancer invasive front associated with infiltrative growth pattern and the risk of disease recurrence. Heterogeneous EGFR GCN increase predicted benefit from EGFR-targeted antibodies, also in the chemorefractory patient population. In summary, high securin and ALDH1 protein expression independently relate to poor outcome in subgroups of rectal cancer patients, potentially because of resistance to conventional chemotherapeutics. Heterogeneous increase in EGFR GCN was validated to be a promising predictive factor in the treatment of metastatic CRC.

Optimization of immunofluorescence protocols for detection of biomarkers in colorectal and breast cancer tissues

Cancer remains an undetermined question for modern medicine. Every year millions of people ranging from children to adult die since the modern treatment is unable to meet the challenge. Research must continue in the area of new biomarkers for tumors. Molecular biology has evolved during last years; however, this knowledge has not been applied into the medicine. Biological findings should be used to improve diagnostics and treatment modalities. In this thesis, human formalin-fixed paraffin embedded colorectal and breast cancer samples were used to optimize the double immunofluorescence staining protocol. Also, immunohistochemistry was performed in order to visualize expression patterns of each biomarker. Concerning double immunofluorescence, feasibility of primary antibodies raised in different and same host species was also tested. Finally, established methods for simultaneous multicolor immunofluorescence imaging of formalin-fixed paraffin embedded specimens were applied for the detection of pairs of potential biomarkers of colorectal cancer (EGFR, pmTOR, pAKT, Vimentin, Cytokeratin Pan, Ezrin, E-cadherin) and breast cancer (Securin, PTTG1IP, Cleaved caspase 3, ki67).

Surgical Treatment of Colorectal Cancer. Controversial Issues

Aims: This study was carried out to evaluate surgical treatment of colorectal cancer (CRC) with special interest in present status and controversial issues: stenting as a palliative procedure for metastasized CRC (I), duration of thromboprophylaxis after the surgical treatment of CRC (II), treatment of the increasing population of elderly people (III) and the quality of life (QoL) after surgery for rectal cancer with special reference to pelvic floor dysfunction (IV). Materials and methods: The material consisted of patients with CRC operated on at Turku University Hospital between 2003 and 2008. In study II the data was collected retrospectively from electronic archives. In other studies the follow-up data was collected at postoperative control visits. In study IV the RAND-36 standardized questionnaire and additional questions assessing urinary, sexual and anorectal dysfunction were used. Results: The results of the current study showed that self-expanding metallic stents provided an alternative to palliative surgery in the treatment of obstructive CRC. Low molecular heparin given s.c. for a median of 11 days until hospital discharge seemed to provide sufficient thromboprophylaxis after surgery. With preoperative selection elderly patients with rectal cancer were suitable for major surgery for rectal cancer with morbidity and mortality rates comparable to those in younger patients. There was no difference between preoperative and one year postoperative general QoL for operated rectal cancer patients. Postoperative pelvic dysfunction was associated with an impaired QoL in some dimensions. Conclusions: Many individual factors regarding the patient and the disease must be taken into account when making treatment decisions in CRC to ensure successful treatment of CRC, patient satisfaction and QoL.

Treatment-related renal side-effects in pediatric cancer patients

Background: The long-term side-effects of cancer treatments are of growing importance, since the number of pediatric cancer survivors has considerably increased. Renal side-effects should be noted early to prevent further deterioration. Renal dysfunction may also develop long after cancer treatment. Easy and reliable methods for assessing renal function are needed. Aims: The aims were to find the mechanisms behind methotrexate-induced renal damage by studying renal tubular cells (LLC-PK1cells), and to evaluate the usefulness of laboratory tests in assessing glomerular function in pediatric cancer patients by comparing an isotope clearance method with alternative methods. The aim was also to study the long-term effects of bone marrow transplantation (BMT) and high-dose methotrexate (HD-MTX) treatment in renal function. Results: Methotrexate induced time-dependent renal tubular cell swelling and cell death. In patients treated with HD-MTX a significant decrease in GFR was noted after a follow-up time of one to ten years. One year after BMTthe GFR was reduced, especially in patients treated with total body irradiation (TBI). GFR recovered slightly but remained stable thereafter. In glomerular function assessment the serum cystatin C (cysC) concentration showed a significant association with GFR measured by the isotope method. Conclusions: Methotrexate induced acute damage in renal tubular cells. In assessing GFR the isotope method still remains the method of choice, but the assay of cystatin C was the most reliable of other alternatives. Long-term follow-up of renal function is needed in BMT patients and patients treated with HD-MTX.

Hypoxia-associated biomarkers in rectal cancer treated by preoperative radiotherapy or chemoradiotherapy

Hypoksiaan liittyvät biologiset merkkiaineet leikkausta edeltävällä sädehoidolla tai kemosädehoidolla hoidetussa peräsuolisyövässä Peräsuolensyöpä on yleinen pahanlaatuinen kasvain. Leikkausta edeltävä sädehoito annetaan yleensä T3-T4-kasvaimille. Tutkimuksella pyrittiin selvittämään, voidaanko kasvaimen hapenpuutteeseen liittyvillä biologisilla merkkiaineilla arvioida peräsuolisyövän ennustetta leikkausta edeltävän sädehoidon tai kemosädehoidon jälkeen. Tällaisia merkkiaineita ovat hapenpuutteen vaikutuksesta aktivoituva HIF-1alfa hiilihappoanhydraasi IX (CA IX), sokerin kuljetukseen solussa osallistuva GLUT-1 sekä solun tukirankaproteiini ezrin. Tutkimukseen otettiin 178 potilasta, jotka olivat saaneet ennen leikkausta lyhyen (n=77) tai pitkän sädehoidon (n=10), pitkän sädehoidon ja solunsalpaajahoidon (n=37) tai ei mitään hoitoa (n=54). Lisäksi osalta leikkausta edeltävää sädehoitoa saaneelta potilaalta tutkittiin hoitoja edeltävät, diagnostiset näytteet (n=80). Tutkimuksessa käytettiin immunehistokemiallisia värjäysmenetelmiä. Kasvaimen regressiota (TRG) arvioitiin pitkän sädehoidon jälkeisistä näytteistä. Leikkausnäytteissä negatiivinen/heikko CA IX intensiteetti liittyi sekä pidempään tautispesifiseen (p=0.034) että tautivapaaseen elinaikaan (p=0.003) ja pitkän sädehoidon jälkeen HIF-1alfa-negatiivisuus pidempään tautispesifiseen (p=0.001) sekä negatiivinen/heikko GLUT-1 pidempään tautivapaaseen elinaikaan (p=0.066). Voimakas ezrin-ilmentymä diagnostisissa näytteissä liittyi lyhyempään tautivapaaseen ja tautispesifiseen (p=0.027 ja p=0.002) ennusteeseen. Monimuuttuja-analyysissä vahva CA IX intensiteetti leikkausnäytteissä ennusti itsenäisesti huonompaa tautivapaata ja tautispesifistä selviytymistä. Erinomainen TRG liittyi negatiiviseen/heikkoon CA IX- (p=0.057), ezrin- (p=0.012) ja GLUT-1 -ilmentymään (p=0.013) leikkausnäytteissä. Kun kaikki neljä merkkiainetta analysoitiin yhdessä monimuuttuja-analyysissä, CA IX intensiteetti leikkausnäytteissä ennusti itsenäisesti tautispesifistä elinaikaa. Voimakas CA IX-ilmentymä leikkausnäytteissä ja positiivinen HIF-1alfa- ja vahva GLUT-1-ilmentymä pitkän sädehoidon jälkeisissä leikkausnäytteissä sekä vahva ezrin-ilmentymä diagnostisissa näytteissä liittyivät epäsuotuisaan ennusteeseen. Monimuuttujaanalyysissä kohtalainen/voimakas CA IX intensiteetti leikkausnäytteissä ennusti itsenäisesti huonompaa tautivapaata ja tautispesifistä elinaikaa. CA IX on vahva biologinen merkkiaine peräsuolisyövässä.

Functional Study of Oncogenic Transcription Factor ERG and its Signaling in Prostate Cancer

TMPRSS2–ERG is the most frequent type of genomic rearrangement present in prostate tumors, in which the 5- prime region of the TMPRSS2 gene is fused to the ERG oncogene. TMPRSS2, containing androgen response elements (AREs), is regulated by androgens in the prostate. The truncated TMPRSS2-ERG fusion transcript is overexpressed in half of the prostate cancer patients. The formation of TMPRSS2-ERG transcript is an early event in prostate carcinogenesis and previous in vivo and in vitro studies have shown ectopic ERG expression to be associated with increased cell invasion. However, the molecular function of ERG and its role in cell signaling is poorly understood. In this study, genomic rearrangement of ERG with TMPRSS2 was studied by using comparative genomic hybridization (CGH) in prostate cancer samples. The biological processes associated with the ERG oncogene expression in prostate epithelial cells were studied, and the results were compared with findings observed in clinical prostate tumor samples. The gene expression data indicated that increased WNT signaling and loss of cell adhesion were a characteristic of TMPRSS2- ERG fusion positive prostate tumor samples. Up- regulation of WNT pathway genes were present in ERG positive prostate tumors, with frizzled receptor 4 (FZD4) presenting with the highest association with ERG overexpression, as verified by quantitative reverse transcription-PCR, immunostaining, and immunoblotting in TMPRSS2-ERG positive VCaP prostate cancer cells. Furthermore, ERG and FZD4 silencing increased cell adhesion by inducing active β1-integrin and E-cadherin expression in VCaP cells. Furthermore, we found a novel inhibitor, 4-(chloromethyl) benzoyl chloride which inhibited the WNT signaling and induced similar phenotypic effects as observed after ERG or FZD4 down regulation in VCaP cells. In conclusion, this work deepens our understanding on the complex oncogenic mechanisms of ERG in prostate cancer that may help in developing drugs against TMPRSS2-ERG positive tumors.

Identification of novel regulators for tumor progression in breast cancer

Breast cancer is the most frequent solid tumor among women and the leading cause of cancer related death in women worldwide. The prognosis of breast cancer patients is tightly correlated with the degree of spread beyond the primary tumor. In this thesis, the aim was to identify novel regulators of tumor progression in breast cancer as well as to get insights into the molecular mechanisms of breast cancer progression and metastasis. First, the role of phospholipid remodeling genes and enzymes important for breast cancer progression was studied in breast cancer samples as well as in cultured breast cancer cells. Tumor samples displayed increased de novo synthesized fatty acids especially in aggressive breast cancer. Furthermore, RNAi mediated cell based assays implicated several target genes critical for breast cancer cell proliferation and survival. Second, the role of arachidonic acid pathway members 15-hydroxyprostaglandin dehydrogenase (HPGD) and phospholipase A2 group VII (PLA2G7) in tumorigenesis associated processes was explored in metastatic breast cancer cells. Both targets were found to contribute to epithelial-mesenchymal transition related processes. Third, a high-throughput RNAi lysate microarray screen was utilized to identify novel vimentin expression regulating genes. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was found to promote cellular features connected with metastatic disease, thus implicating MTHFD2 as a potential drug target to block breast cancer cell migration and invasion. Taken together, this study identified several putative targets for breast cancer therapy. In addition, these results provide novel information about the mechanisms and factors underlying breast cancer progression.

Identification of Epigenetic Targets in Prostate Cancer for Therapeutic Development

Recurrent castration resistant prostate cancer remains a challenge for cancer therapies and novel treatment options in addition to current anti-androgen and mitosis inhibitors are needed. Aberrations in epigenetic enzymes and chromatin binding proteins have been linked to prostate cancer and they may form a novel class of drug targets in the future. In this thesis we systematically evaluated the epigenenome as a prostate cancer drug target. We functionally silenced 615 known and putative epigenetically active protein coding genes in prostate cancer cell lines using high throughput RNAi screening and evaluated the effects on cell proliferation, androgen receptor (AR) expression and histone patterns. Histone deacetylases (HDACs) were found to regulate AR expression. Furthermore, HDAC inhibitors reduced AR signaling and inhibited synergistically with androgen deprivation prostate cancer cell proliferation. In particular, TMPRSS2- EGR fusion gene positive prostate cancer cell lines were sensitive to combined HDAC and AR inhibition, which may partly be related to the dependency of a fusion gene induced epigenetic pathway. Histone demethylases (HDMs) were identified to regulate prostate cancer cell line proliferation. We discovered a novel histone JmjC-domain histone demethylase PHF8 to be highly expressed in high grade prostate cancers and mediate cell proliferation, migration and invasion in in vitro models. Additionally, we explored novel HDM inhibitor chemical structures using virtual screening methods. The structures best fitting to the active pocket of KDM4A were tested for enzyme inhibition and prostate cancer cell proliferation activity in vitro. In conclusion, our results show that prostate cancer may efficiently be targeted with combined AR and HDAC inhibition which is also currently being tested in clinical trials. HDMs were identified as another feasible novel drug target class. Future studies in representative animal models and development of specific inhibitors may reveal HDMs full potential in prostate cancer therapy

The Role of an Oncoprotein CIP2A in Breast Cancer

Cancerous inhibitor of PP2A (CIP2A) is an oncoprotein expressed in several human cancer types. Previously, CIP2A has been shown to promote proliferation of cancer cells. Mechanistically, CIP2A is known to inhibit activity of a tumor suppressor protein phosphatase 2A (PP2A) towards an oncoprotein MYC, further stabilizing MYC in human cancer. However, the molecular mechanisms how CIP2A expression is induced during cellular transformation are not well known. Also, expression, functional role and clinical relevance of CIP2A in breast cancer had not been studied before. The results of this PhD thesis work demonstrate that CIP2A is highly expressed in human breast cancer, and that high expression of CIP2A in tumors is a poor prognostic factor in a subset of breast cancer patients. CIP2A expression correlates with inactivating mutations of tumor suppressor p53 in human cancer. Notably, we demonstrate that p53 inactivation up-regulates CIP2A expression via increased expression of an oncogenic transcription factor E2F1. Moreover, CIP2A promotes expression of E2F1, and this novel positive feedback loop between E2F1 and CIP2A is demonstrated to regulate sensitivity to both p53-dependent and -independent senescence induction in breast cancer cells. Importantly, in a CIP2A deficient breast cancer mouse model, abrogation of CIP2A attenuates mammary tumor formation and progression with features of E2F1 inhibition and induction of senescence. Furthermore, we demonstrate that CIP2A expression defines the cellular response to a senescence-inducing chemotherapy in breast cancer. Taken together, these results demonstrate that CIP2A is an essential promoter of breast cancer tumor growth by inhibiting senescence. Finally, this study implicates inhibition of CIP2A as a promising therapy target for breast cancer.

Prognostic Factors In Breast Cancer. With Special Reference to Cyclins A, B1, D1 and E, MMP-1 and Decorin

Breast cancer is a highly heterogenous malignancy, which despite of the similar histological type shows different clinical behaviour and response to therapy. Prognostic factors are used to estimate the risk for recurrence and the likelihood of treatment effectiveness. Because breast cancer is one of the most common causes of cancer death in women worldwide, identification of new prognostic markers are needed to develop more specific and targeted therapies. Cancer is caused by uncontrolled cell proliferation. The cell cycle is controlled by specific proteins, which are known as cyclins. They function at important checkpoints by activating cyclin-dependent kinase enzymes. Overexpression of different cyclins has been linked to several cancer types and altered expression of cyclins A, B1, D1 and E has been associated with poor survival. Little is known about the combined expression of cyclins in relation to the tumour grade, breast cancer subtype and other known prognostic factors. In this study cyclins A, B1 and E were shown to correlate with histological grade, Ki-67 and HER2 expression. Overexpression of cyclin D1 correlated with receptor status and non-basal breast cancer suggesting that cyclin D1 might be a marker of good prognosis. Proteolysis in the surrounding tumour stroma is increased during cancer development. Matrix metalloproteinases (MMPs) are proteolytic enzymes that are capable of degrading extracellular matrix proteins. Increased expression and activation of several MMPs have been found in many cancers and MMPs appear to be important regulators of invasion and metastasis. In this study MMP-1 expression was analysed in breast cancer epithelial cells and in cancer associated stromal cells. MMP-1 expression by breast cancer epithelial cells was found to carry an independent prognostic value as did Ki-67 and bcl-2. The results suggest that in addition to stromal cells MMP-1 expression in tumour cells control breast cancer progression. Decorin is a small proteoglycan and an important component of the extracellular matrix. Decorin has been shown to inhibit growth of tumour cells and reduced decorin expression is associated with a poor prognosis in several cancer types. There has been some suspicion wheather different cancer cells express decorin. In this study decorin expression was shown to localize only in the cells of the original stroma, while breast cancer epithelial cells were negative for decorin expression. However, transduction of decorin in decorin-negative human breast cancer cells markedly modulated the growth pattern of these cells. This study provides evidence that targeted decorin transduction to breast cancer cells could be used as a novel adjuvant therapy in breast malignancies.

Collagen binding integrins and cancer testis antigens in prostate cancer and melanoma

Camilla Pelo Collagen Binding Integrins and Cancer Testis Antigens in Prostate Cancer and Melanoma Department of Biochemistry, MediCity Research Laboratory, University of Turku, Finland Annales Universitatis Turkuensis, Painosalama Oy, Turku, Finland 2016 ABSTRACT Prostate cancer is the second most common cancer in men worldwide. The incidence of melanoma, in turn, is increasing faster than any other cancer incidences. In Finland, more than 5000 prostate cancer and 1200 new melanoma cases are diagnosed each year. One approach to further understand the cellular processes involved in prostate cancer and melanoma is to gain better knowledge about alterations in gene expression and their potential impact on the progression of the diseases. This thesis is focused on expression studies in two gene families; integrins and cancer testis antigens (CT antigens), in human prostate adenocarcinoma and advanced human melanoma. Integrins are heterodimeric transmembrane receptors which regulate many important cellular processes such as cell proliferation, migration and survival. CT antigens are frequently expressed in different types of cancers, but are only expressed in testis in healthy individuals. CT antigens are also highly immunogenic proteins. Due to the properties mentioned above, integrins and CT antigens can function as target molecules for the development of cancer diagnostics and drugs. One of the main purposes of this thesis was to study the expression of the four collagen binding integrins α1β1, α2β1, α10β1, α11β1 and the cancer testis antigen 16 (CT16) in cancer cell lines and human tissues of prostate cancer and metastatic melanoma. Additional aims included studies on the biological role of CT16 and the abundance of CT16 in sera of advanced melanoma patients. The prognostic and diagnostic significance of CT16 and the collagen binding integrins were also evaluated. Expression studies on collagen binding integrins and the CT antigen CT16 in melanoma and prostate cancer were limited and the biological role of CT16 was unknown. In this thesis, the expression levels of α2β1 and α11β1 were found to be significantly altered in prostate cancer tissues. Integrin α2β1 decreased gradually during disease progression while α11 was elevated in prostate carcinoma compared to healthy tissues. In advanced melanoma, enhanced levels of α2 were associated with a significant shorter overall survival in advanced melanoma. In this thesis, CT16 was identified as a frequently expressed melanoma CT antigen with an anti-apoptotic function. To conclude, this thesis presents α2β1 and CT16, as potential and promising biomarkers for advanced melanoma. This thesis reports also the first functional study of CT16. Keywords: Collagen binding integrins, α1β1, α2β1, α10β1, α11β1, Cancer Testis antigens, CT16, melanoma, prostate cancer, expression