Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans: 2. Biochemical analyses

Platelet endothelial cell adhesion molecule 1 (PECAM-1) (CD31), a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules with six Ig-like domains, has a range of functions, notably its contributions to leukocyte extravasation during inflammation and in maintaining vascular endothelial integrity. Although PECAM-1 is known to mediate cell adhesion by homophilic binding via domain 1, a number of PECAM-1 heterophilic ligands have been proposed. Here, the possibility that heparin and heparan sulfate (HS) are ligands for PECAM-1 was reinvestigated. The extracellular domain of PECAM-1 was expressed first as a fusion protein with the Fc region of human IgG1 fused to domain 6 and second with an N-terminal Flag tag on domain 1 (Flag-PECAM-1). Both proteins bound heparin immobilized on a biosensor chip in surface plasmon resonance (SPR) binding experiments. Binding was pH-sensitive but is easily measured at slightly acidic pH. A series of PECAM-1 domain deletions, prepared in both expression systems, were tested for heparin binding. This revealed that the main heparin-binding site required both domains 2 and 3. Flag-PECAM-1 and a Flag protein containing domains 1-3 bound HS on melanoma cell surfaces, but a Flag protein containing domains 1-2 did not. Heparin oligosaccharides inhibited Flag-PECAM-1 from binding immobilized heparin, with certain structures having greater inhibitory activity than others. Molecular modeling similarly identified the junction of domains 2 and 3 as the heparin-binding site and further revealed the importance of the iduronic acid conformation for binding. PECAM-1 does bind heparin/HS but by a site that is distinct from that required for homophilic binding.

Keeping kids sun safe: exploring parents' beliefs about their young child's sun-protective behaviours

Objectives Melanoma of the skin is the third most commonly diagnosed cancer in Australia. Given the high incidence of sunburn in children and the level of sun protection provided by parents is often infrequent and/or insufficient, this research employed qualitative methodology to examine parents' beliefs about their young child's sun safe behaviour. Methods Parents (N = 21; n = 14 mothers, n = 7 fathers) of children aged 2–5 years participated in focus groups to identify commonly held beliefs about their decision to sun protect their child. Data were analysed using thematic content analysis. Results Parents generally had knowledge of the broad sun safe recommendations; however, the specific details of the recommendations were not always known. Parents reported adopting a range of sun-protective measures for their child, which depended on the time of year. A range of advantages (e.g. reducing the risk of skin cancer, developing good habits early and parental peace of mind), disadvantages (e.g. false sense of safety and preventing vitamin D absorption), barriers (e.g. child refusal) and facilitators (e.g. routine and accessibility) to performing sun safe practices were identified. Normative pressures and expectations also affected parents' motivation to be sun safe for their child. Conclusions These identified beliefs can be used to inform interventions to improve sun safe behaviours in young children who reside in a region that has the highest skin cancer incidence in the world.

Long-term clinical outcome after liver transplantation

With transplant rejection rendered a minor concern and survival rates after liver transplantation (LT) steadily improving, long-term complications are attracting more attention. Current immunosuppressive therapies, together with other factors, are accompanied by considerable long-term toxicity, which clinically manifests as renal dysfunction, high risk for cardiovascular disease, and cancer. This thesis investigates the incidence, causes, and risk factors for such renal dysfunction, cardiovascular risk, and cancer after LT. Long-term effects of LT are further addressed by surveying the quality of life and employment status of LT recipients. The consecutive patients included had undergone LT at Helsinki University Hospital from 1982 onwards. Data regarding renal function – creatinine and estimated glomerular filtration rate (GFR) – were recorded before and repeatedly after LT in 396 patients. The presence of hypertension, dyslipidemia, diabetes, impaired fasting glucose, and overweight/obesity before and 5 years after LT was determined among 77 patients transplanted for acute liver failure. The entire cohort of LT patients (540 patients), including both children and adults, was linked with the Finnish Cancer Registry, and numbers of cancers observed were compared to site-specific expected numbers based on national cancer incidence rates stratified by age, gender, and calendar time. Health-related quality of life (HRQoL), measured by the 15D instrument, and employment status were surveyed among all adult patients alive in 2007 (401 patients). The response rate was 89%. Posttransplant cardiovascular risk factor prevalence and HRQoL were compared with that in the age- and gender-matched Finnish general population. The cumulative risk for chronic kidney disease increased from 10% at 5 years to 16% at 10 years following LT. GFR up to 10 years after LT could be predicted by the GFR at 1 year. In patients transplanted for chronic liver disease, a moderate correlation of pretransplant GFR with later GFR was also evident, whereas in acute liver failure patients after LT, even severe pretransplant renal dysfunction often recovered. By 5 years after LT, 71% of acute liver failure patients were receiving antihypertensive medications, 61% were exhibiting dyslipidemia, 10% were diabetic, 32% were overweight, and 13% obese. Compared with the general population, only hypertension displayed a significantly elevated prevalence among patients – 2.7-fold – whereas patients exhibited 30% less dyslipidemia and 71% less impaired fasting glucose. The cumulative incidence of cancer was 5% at 5 years and 13% at 10. Compared with the general population, patients were subject to a 2.6-fold cancer risk, with non-melanoma skin cancer (standardized incidence ratio, SIR, 38.5) and non-Hodgkin lymphoma (SIR 13.9) being the predominant malignancies. Non-Hodgkin lymphoma was associated with male gender, young age, and the immediate posttransplant period, whereas old age and antibody induction therapy raised skin-cancer risk. HRQoL deviated clinically unimportantly from the values in the general population, but significant deficits among patients were evident in some physical domains. HRQoL did not seem to decrease with longer follow-up. Although 87% of patients reported improved working capacity, data on return to working life showed marked age-dependency: Among patients aged less than 40 at LT, 70 to 80% returned to work, among those aged 40 to 50, 55%, and among those above 50, 15% to 28%. The most common cause for unemployment was early retirement before LT. Those patients employed exhibited better HRQoL than those unemployed. In conclusion, although renal impairment, hypertension, and cancer are evidently common after LT and increase with time, patients’ quality of life remains comparable with that of the general population.

Matrix metalloproteinases as biomarkers in premalignant and malignant tumors of the human skin

The incidence of non-melanoma skin cancer is increasing worldwide. Basal cell carcinoma followed by squamous cell carcinoma and malignant melanoma are the most frequent skin tumors. Immunosuppressed patients have an increased risk of neoplasia, of which non-melanoma skin cancer is the most common. Matrix metalloproteinases (MMPs) are proteolytic enzymes that collectively are capable of degrading virtually all components of the extracellular matrix. MMPs can also process substrates distinct from extracellular matrix proteins and influence cell proliferation, differentiation, angiogenesis, and apoptosis. MMP activity is regulated by their natural inhibitors, tissue inhibitors of metallopro-teinases (TIMPs). In this study, the expression patterns of MMPs, TIMPs, and certain cancer-related molecules were investigated in premalignant and malignant lesions of the human skin. As methods were used immunohistochemisty, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR) from the cell cultures. Our aim was to evaluate the expression pattern of MMPs in extramammary Paget's disease in order to find markers for more advanced tumors, as well as to shed light on the origin of this rare neoplasm. Novel MMPs -21, -26, and -28 were studied in melanoma cell culture, in primary cutaneous melanomas, and their sentinel nodes. The MMP expression profile in keratoacanthomas and well-differentiated squamous cell carcinomas was analyzed to find markers to differentiate benign keratinocyte hyperproliferation from malignantly transformed cells. Squamous cell carcinomas of immunosuppressed organ transplant recipients were compared to squamous cell carcinomas of matched immunocompetent controls to investigate the factors explaining their more aggressive nature. We found that MMP-7 and -19 proteins are abundant in extramammary Paget's disease and that their presence may predict an underlying adenocarcinoma in these patients. In melanomas, MMP-21 was upregulated in early phases of melanoma progression, but disappeared from the more aggressive tumors with lymph node metastases. The presence of MMP-13 in primary melanomas and lymph node metastases may relate to more aggressive disease. In keratoacanthomas, the expression of MMP-7 and -9 is rare and therefore should raise a suspicion of well-differentiated squamous cell carcinomas. Furthermore, MMP-19 and p16 were observed in benign keratinocyte hyperproliferation of keratoacanthomas, whereas they were generally lost from malignant keratinocytes of SCCs. MMP-26 staining was significantly stronger in squamous cell carcinomas and Bowen s disease samples of organ transplant recipients and it may contribute to the more aggressive nature of squamous cell carcinomas in immunosuppressed patients. In addition, the staining for MMP-9 was significantly stronger in macrophages surrounding the tumors of the immunocompetent group and in neutrophils of those patients on cyclosporin medication. In conclusion, based on our studies, MMP-7 and -19 might serve as biomarkers for more aggressive extramammary Paget's disease and MMP-21 for malignant transformation of melanocytes. MMP -7, -9, and -26, however, could play an important role in the pathobiology of keratinocyte derived malignancies.

The Risk of Cancer Associated with Immunosuppressive Therapy for Skin Diseases

The possible carcinogenic risk of immunosuppressive therapies is an important issue in everyday clinical practise. Carcinogenesis is a slow multi step procedure, thus a long latency period is needed before cancer develops. PUVA therapy is used for many skin diseases including psoriasis, early stage cutaneous T cell lymphoma, atopic dermatitis, palmoplantar pustulosis and chronic eczema. There has been concern about the increased melanoma risk associated to PUVA therapy, which has previously been associated with an increased risk on non-melanoma skin cancer, especially squamous cell carcinoma. The increased risk of basal cell carcinoma (BCC) is also documented but it is modest compared to squamous cell carcinoma (SCC). This thesis evaluated melanoma and noncutaneous cancer risk associated to PUVA, and the persistence of nonmelanoma cancer risk after the cessation of PUVA treatment. Also, the influence of photochemotherapy to the development of secondary cancers in cutaneous T cell lymphoma and the role of short term cyclosporine in later cancer development in inflammatory skin diseases were evaluated. The first three studies were performed on psoriasis patients. The risk of melanoma started to increase 15 years after the first treatment with PUVA. The risk was highest among persons who had received over 250 treatments compared to those under 250 treatments. In noncutaneous cancer, the overall risk was not increased (RR=1.08,95% CI=0.93-1.24), but significant increases in risk were found in thyroid cancer, breast cancer and in central nervous system neoplasms. These cancers were not associated to PUVA. The increased risk of SCC was associated to high cumulative UVA exposure in the PUVA regimen. The patients with high risk had no substantial exposure to other carcinogens. In BCC there was a similar but more modest tendency. In the two other studies, the risk of all secondary cancers (SIR) in CTCL patients was 1.4 (95% CI=1.0-1.9). In separate sites, the risk of lung cancer, Hodgkin and non-Hodgkin lymphomas were increased. PUVA seemed not to contribute to any extent to the appearance of these cancers. The carcinogenity of short-term cyclosporine was evaluated in inflammatory skin diseases. No increased risk for any type of cancer including the skin cancers was detected. To conclude, our studies confirm the increased skin cancer risk related to PUVA treatment in psoriasis patients. In clinical practice, this has led to a close and permanent follow-up of patients treated with PUVA. In CTCL patients, PUVA treatment did not contribute to the development of secondary cancers. We could not detect any increase in the risk of cancer in patients treated with short term cyclosporine, unlike in organ transplant patients under such long-term therapy.

Iodine Brachytherapy for Large Uveal Melanomas

Uveal melanoma is the most common primary intraocular malignancy in adults. Vision in the affected eye is threatened by both the tumor and side-effects from the treatments currently available. Poor prognosis for saving vision increases with tumor size and, consequently, enucleation has been the treatment of choice for large uveal melanomas in most centers. However, increasing evidence suggests that no survival benefit is gained (nor lost) by enucleation as compared to eye-conserving methods. The Helsinki University Eye Hospital has since 1990 offered episcleral iodine-125 plaque brachytherapy (IBT) for all patients unwilling to undergo enucleation for a large uveal melanoma. The primary aim of this study was to assess survival, local tumor recurrence and preservation of the eye and vision after IBT in a population-based series of 97 patients with uveal melanomas classified as large by the Collaborative Ocular Melanoma Study (COMS) criteria. Further aims included reporting the incidence of side-effects and assessing the role of intraocular dose distribution and clinical risk factors in their development. Finally, means to improve the current treatment were investigated by using computer models to compare existing plaques with collimating ones and by comparing the outcome of a subgroup of 54 IBT patients with very thick tumors with 33 patients with similarly-sized tumors managed with transscleral local resection (TSR) in Liverpool, United Kingdom. Kaplan-Meier estimates of all-cause and melanoma-specific survival at 5 years after IBT were 62% and 65%, respectively, and visually comparable with the survival experience of patients reported after enucleation by the COMS. Local recurrence developed in 6% of eyes and 84% of eyes were conserved at 5 years. Visual prognosis was guarded with 11% avoiding loss of 20/70 vision and 26% avoiding loss of 20/400 vision in the tumor eye at 2 years. Large tumor height and short distance from the posterior pole were independently associated with loss of vision. Using cumulative incidence analysis to account for competing risks, such as enucleation and metastatic death, the 5-year incidence of cataract after IBT was 79%, glaucoma 60%, optic neuropathy 46%, maculopathy 52%, persistent or recurring retinal detachment (RD) 25%, and vitreous hemorrhage 36%. In multivariate competing risks regression models, increasing tumor height was associated with cataract, iris neovascularization and RD. Maculopathy and optic neuropathy were associated with distance from the tumor to the respective structure. Median doses to the tumor apex, macula and optic disc were 81 Gy (range, 40-158), 79 Gy (range, 12-632), and 83 Gy (range, 10-377), respectively. Dose to the optic disc was independently associated with optic neuropathy, and both dose to the optic disc and dose to the macula predicted vision loss after IBT. Simulated treatment using collimating plaques resulted in clinically meaningful reduction in both optic disc (median reduction, 30 Gy) and macular (median reduction, 36 Gy) doses as compared to the actual treatment with standard plaques. In the subgroup of patients with uveal melanomas classified as large because of tumor height, cumulative incidence analysis revealed that while long-term preservation of 20/70 vision was rare after both IBT and TSR, preservation of 20/400 vision was better after TSR (32% vs. 5% at 5 years). In multivariate logistic regression models, TSR was independently associated with better preservation of 20/400 vision (OR 0.03 at 2 years, P=0.005) No cases of secondary glaucoma were observed after TSR and optic neuropathy was rare. However, local tumor recurrence was more common after TSR than it was after IBT (Cumulative incidence 41% vs. 7% at 5 years, respectively). In terms of survival, IBT seems to be a safe alternative to enucleation in managing large uveal melanomas. Local tumor control is no worse than with medium-sized tumors and the chances of avoiding secondary enucleation are good. Unfortunately, side-effects from radiotherapy are frequent, especially in thick tumors, and long-term prognosis of saving vision is consequently guarded. Some complications can be limited by using collimating plaques and by managing uveal melanomas that are large because of tumor height with TSR instead of IBT. However, the patient must be willing to accept a substantial risk of local tumor recurrence after TSR and it is best suited for cases in which the preservation of vision in the tumor eye is critical.

Digital Photography in the Diagnosis and Follow-up of Ocular Diseases

Purpose: The aim of the present study was to develop and test new digital imaging equipment and methods for diagnosis and follow-up of ocular diseases. Methods: The whole material comprised 398 subjects (469 examined eyes), including 241 patients with melanocytic choroidal tumours, 56 patients with melanocytic iris tumours, 42 patients with diabetes, a 52-year old patient with chronic phase of VKH disease, a 30-year old patient with an old blunt eye injury, and 57 normal healthy subjects. Digital 50° (Topcon TRC 50 IA) and 45° (Canon CR6-45NM) fundus cameras, a new handheld digital colour videocamera for eye examinations (MediTell), a new subtraction method using the Topcon Image Net Program (Topcon corporation, Tokyo, Japan), a new method for digital IRT imaging of the iris we developed, and Zeiss photoslitlamp with a digital camera body were used for digital imaging. Results: Digital 50° red-free imaging had a sensitivity of 97.7% and two-field 45° and 50° colour imaging a sensitivity of 88.9-94%. The specificity of the digital 45°-50° imaging modalities was 98.9-100% versus the reference standard and ungradeable images that were 1.2-1.6%. By using the handheld digital colour video camera only, the optic disc and central fundus located inside 20° from the fovea could be recorded with a sensitivity of 6.9% for detection of at least mild NPDR when compared with the reference standard. Comparative use of digital colour, red-free, and red light imaging showed 85.7% sensitivity, 99% specificity, and 98.2 % exact agreement versus the reference standard in differentiation of small choroidal melanoma from pseudomelanoma. The new subtraction method showed growth in four of 94 melanocytic tumours (4.3%) during a mean ±SD follow-up of 23 ± 11 months. The new digital IRT imaging of the iris showed the sphincter muscle and radial contraction folds of Schwalbe in the pupillary zone and radial structural folds of Schwalbe and circular contraction furrows in the ciliary zone of the iris. The 52-year-old patient with a chronic phase of VKH disease showed extensive atrophy and occasional pigment clumps in the iris stroma, detachment of the ciliary body with severe ocular hypotony, and shallow retinal detachment of the posterior pole in both eyes. Infrared transillumination imaging and fluorescein angiographic findings of the iris showed that IR translucence (p=0.53), complete masking of fluorescence (p=0.69), presence of disorganized vessels (p=0.32), and fluorescein leakage (p=1.0) at the site of the lesion did not differentiate an iris nevus from a melanoma. Conclusions: Digital 50° red-free and two-field 50° or 45° colour imaging were suitable for DR screening, whereas the handheld digital video camera did not fulfill the needs of DR screening. Comparative use of digital colour, red-free and red light imaging was a suitable method in the differentiation of small choroidal melanoma from different pseudomelanomas. The subtraction method may reveal early growth of the melanocytic choroidal tumours. Digital IRT imaging may be used to study changes of the stroma and posterior surface of the iris in various diseases of the uvea. It contributed to the revealment of iris atrophy and serous detachment of the ciliary body with ocular hypotony together with the shallow retinal detachment of the posterior pole as new findings of the chronic phase of VKH disease. Infrared translucence and angiographic findings are useful in differential diagnosis of melanocytic iris tumours, but they cannot be used to determine if the lesion is benign or malignant.

Clinician perspective on molecular profiling of non-small-cell lung cancer

The article by Meric-Bernstam et al1 that was recently published in Journal of Clinical Oncology raises important questions about the clinical application of large-scale genomic testing. We congratulate the authors for this ambitious study, which successfully profiled 2,000 consecutive patients with advanced cancer. The next-generation sequencing (NGS) platform was used for 1,749 of 2,000 patients (87.5%). Of 789 patients with potentially actionable mutations, 83 (11%, or 4% of screened population) were enrolled in a genomically matched clinical study. As the editorial2 accompanying the article by Meric-Bernstam et al1 pointed out, the 4% figure, albeit disappointing, may be an underestimate because cancers such as lung adenocarcinoma and melanoma, for which ≥ 50% of patients have actionable mutations, were under-represented. ...

Stimulating immune responses to fight cancer: Basic biology and mechanisms

Chronic inflammation is now recognized as a major cause of malignant disease. In concert with various mechanisms (including DNA instability), hypoxia and activation of inflammatory bioactive lipid pathways and pro-inflammatory cytokines open the doorway to malignant transformation and proliferation, angiogenesis, and metastasis in many cancers. A balance between stimulatory and inhibitory signals regulates the immune response to cancer. These include inhibitory checkpoints that modulate the extent and duration of the immune response and may be activated by tumor cells. This contributes to immune resistance, especially against tumor antigen-specific T-cells. Targeting these checkpoints is an evolving approach to cancer immunotherapy, designed to foster an immune response. The current focus of these trials is on the programmed cell death protein 1 (PD-1) receptor and its ligands (PD-L1, PD-L2) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Researchers have developed anti-PD-1 and anti-PDL-1 antibodies that interfere with the ligands and receptor and allow the tumor cell to be recognized and attacked by tumor-infiltrating T-cells. These are currently being studied in lung cancer. Likewise, CTLA-4 inhibitors, which have had success treating advanced melanoma, are being studied in lung cancer with encouraging results.

Identification of molecules relevant for the invasiveness of fibrosarcomas and melanomas

Cancer is becoming the leading cause of deaths in the world. As 90% of all deaths from cancer are caused by metastasis, discovery of the mechanisms behind cancer cell invasion and metastasis is of utmost importance. Only new effective therapies targeting cancer progression can reduce cancer mortality rates. The aim of this study was to identify molecules that are relevant for tumor cell invasion and spreading in fibrosarcomas and melanomas, and to analyze their potential for cancer biomarkers or therapeutic targets. First, the gene expression changes of normal cells and transformed cells showing high invasiveness, S-adenosylmethionine decarboxylase (AdoMetDC)-transfected murine fibroblasts and human melanoma cells, were studied by microarray analyses. The function of the identified candidate molecules were then studied in detail in these cell lines. Finally, the physiological relevance of the identified changes was studied by immunohistochemical analyses of human sarcoma and melanoma specimens or by a mouse xenograft model. In fibrosarcoma cells, the most remarkable change detected was a dramatic up-regulation of the actin-sequestering molecule thymosin beta 4 (TB4), which was shown to be important for the transformed phenotype of the AdoMetDC-transfected cells (Amdc-s and -as). A sponge toxin latrunculin A, inhibiting the binding of TB4 to actin, was found to selectively inhibit the migration and invasion of these cells. Further, Amdc-s-induced mouse tumors and human high-grade sarcomas were found to show intense TB4 immunostaining. In addition to TB4, integrin subunits alfa 6 and beta 7 (ItgA6 and ItgB7) were found to be up-regulated in Amdc-s and -as cells. ItgA6 was shown to dimerize mainly with ItgB1 in Amdc-s. Inhibition of ItgA6 or ItgB1 function with neutralizing antibodies fully blocked the invasiveness of Amdc-s cells, and importantly also human HT-1080 fibrosarcoma cells, in three-dimensional (3D)-Matrigel mimicking tumor extracellular matrix (ECM). By immunohistochemical analyses, strong staining for ITGA6 was detected in human high-grade fibrosarcomas and other sarcomas, especially at the invasion fronts of the tumors. In the studied melanoma cell lines, the expression levels of the adhesion-related ECM proteins tenascin-C (TN-C), fibronectin (FN), and transforming growth factor beta-induced (TGFBI) were found to be highly up-regulated. By immunohistochemistry, intense TN-C and FN staining was detected in invasive and metastatic melanoma tumors, showing co-localization (together with procollagen-I) in tubular meshworks and channels around the invading melanoma cells. In vitro, TN-C and FN were further found to directly stimulate the migration of melanoma cells in 3D-collagen-I matrix. The third candidate protein, TGFBI, was found to be an anti-adhesive molecule for melanoma cells, and knockdown of its expression in metastatic melanoma cells (TGFBI-KD cells) led to dramatically impaired tumor growth in immunocompromized mice. Interestingly, the control tumors showed intense TGFBI immunostaining in the invasion fronts, showing partial co-localization with the fibrillar FN staining, whereas the small TGFBI-KD cell-induced tumors displayed amorphous, non-fibrillar FN staining. These data suggest an important role for TGFBI in FN fibrillogenesis and melanoma progression. In conclusion, we have identified several invasion-related molecules, which show potential for cancer diagnostic or prognostic markers, or therapeutic targets. Based on our previous and present fibrosarcoma studies, we propose the possibility of using ITGA6 antagonists (affecting tumor cell adhesion) in combination with TB4 inhibitors (affecting tumor cell migration) and cathepsin L inhibitors (affecting the degradation of basement membrane and ECM proteins) for the treatment of fibrosarcomas and other tumors overexpressing these molecules. With melanoma cells, in turn, we point to the importance of three secreted ECM proteins, TN-C, FN, and TGFBI, in melanoma progression. Of these, especially the potential of TN-C as a prognostic melanoma biomarker and TGFBI as a promising therapeutic target molecule are clearly worth additional studies.

Identification of TGFβ signaling, p53, and actin stress fibers as targets of LKB1 tumor suppressor activity

Tumorigenesis is a consequence of inactivating mutations of tumor suppressor genes and activating mutations of proto-oncogenes. Most of the mutations compromise cell autonomous and non-autonomous restrains on cell proliferation by modulating kinase signal transduction pathways. LKB1 is a tumor suppressor kinase whose sporadic mutations are frequently found in non-small cell lung cancer and cervical cancer. Germ-line mutations in the LKB1 gene lead to Peutz-Jeghers syndrome with an increased risk of cancer and development of benign gastrointestinal hamartomatous polyps consisting of hyperproliferative epithelia and prominent stromal stalk composed of smooth muscle cell lineage cells. The tumor suppressive function of LKB1 is possibly mediated by 14 identified LKB1 substrate kinases, whose activation is dependent on the LKB1 kinase complex. The aim of my thesis was to identify cell signaling pathways crucial for tumor suppression by LKB1. Re-introduction of LKB1 expression in the melanoma cell line G361 induces cell cycle arrest. Here we demonstrated that restoring the cytoplasmic LKB1 was sufficient to induce the cell cycle arrest in a tumor suppressor p53 dependent manner. To address the role of LKB1 in gastrointestinal tumor suppression, Lkb1 was deleted specifically in SMC lineage in vivo, which was sufficient to cause Peutz-Jeghers syndrome type polyposis. Studies on primary myofibroblasts lacking Lkb1 suggest that the regulation of TGFβ signaling, actin stress fibers and smooth muscle cell lineage differentiation are candidate mechanisms for tumor suppression by LKB1 in the gastrointestinal stroma. Further studies with LKB1 substrate kinase NUAK2 in HeLa cells indicate that NUAK2 is part of a positive feedback loop by which NUAK2 expression promotes actin stress fiber formation and, reciprocally the induction of actin stress fibers promote NUAK2 expression. Findings in this thesis suggest that p53 and TGFβ signaling pathways are potential mediators of tumor suppression by LKB1. An indication of NUAK2 in the promotion of actin stress fibers suggests that NUAK2 is one possible mediator of LKB1 dependent TGFβ signaling and smooth muscle cell lineage differentiation.

Proteomic Identification of Haptoglobin alpha 2 as a Glioblastoma Serum Biomarker: Implications in Cancer Cell Migration and Tumor Growth

Glioblastoma (GBM; grade IV astrocytoma) is the most malignant and common primary brain tumor in adults. Using combination of 2-DE and MALDI-TOF MS, we analyzed 14 GBM and 6 normal control sera and identified haptoglobin alpha 2 chain as an up-regulated serum protein in GBM patients. GBM-specific up-regulation was confirmed by ELISA based quantitation of haptoglobin (Hp) in the serum of 99 GBM patients as against lower grades (49 grade III/AA; 26 grade II/DA) and 26 normal individuals (p = 0.0001). Further validation using RT-qPCR on an independent set (n = 78) of tumor and normal brain (n = 4) samples and immunohistochemcial staining on a subset (n = 42) of above samples showed increasing levels of transcript and protein with tumor grade and were highest in GBM (p = < 0.0001 and < 0.0001, respectively). Overexpression of Hp either by stable integration of Hp cDNA or exogenous addition of purified Hp to immortalized astrocytes resulted in increased cell migration. RNAi-mediated silencing of Hp in glioma cells decreased cell migration. Further, we demonstrate that both human glioma and mouse melanoma cells overexpressing Hp showed increased tumor growth. Thus, we have identified haptoglobin as a GBM-specific serum marker with a role on glioma tumor growth and migration.

Regulation of S100A2 expression by TGF-beta-induced MEK/ERK signalling and its role in cell migration/invasion

S100A2, an EF hand calcium-binding protein, is a potential biomarker in several cancers and is also a TGF-beta (transforming growth factor-beta)-regulated gene in melanoma and lung cancer cells. However, the mechanism of S100A2 regulation by TGF-beta and its significance in cancer progression remains largely unknown. In the present study we report the mechanism of S100A2 regulation by TGF-beta and its possible role in TGF-beta-mediated tumour promotion. Characterization of the S100A2 promoter revealed an AP-1 (activator protein-1) element at positions -1161 to -1151 as being the most critical factor for the TGF-beta 1 response. Chromatin immunoprecipitation and electrophoretic mobility-shift assays confirmed the functional binding of the AP-1 complex, predominantly JunB, to the S100A2 promoter in response to TGF-beta 1 in HaCaT keratinocytes. JunB overexpression markedly stimulated the S100A2 promoter which was blocked by the dominant-negative JunB and MEK1 MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1] inhibitor, PD98059. Intriguingly, despite the presence of a putative SMAD-binding element, S100A2 regulation by TGF-beta 1 was found to be SMAD3 independent. Interestingly, p53 protein and TGF-beta 1 show synergistic regulation of the S100A2 promoter. Finally, knockdown of S100A2 expression compromised TGF-beta 1-induced cell migration and invasion of Hep3B cells. Together our findings highlight an important link between the TGF-beta 1-induced MAPK and p53 signalling pathways in the regulation of S100A2 expression and pro-tumorigenic actions.

Effect of a natural mutation in the 5 ` untranslated region on the translational control of p53 mRNA

Tumor-suppressor protein p53, the `guardian of the genome', is critical in maintaining cellular homeostasis and genomic stability. Earlier, we have reported the discovery of internal ribosome entry sites (IRESs) within the p53 mRNA that regulate the translation of the full length and its N-terminal-truncated isoform, Delta N-p53. Polypyrimidine tract-binding protein (PTB) is an IRES trans-acting factor that positively regulates the IRES activities of both p53 isoforms by relocating from nucleus to the cytoplasm during stress conditions. Here we have demonstrated the putative contact points of PTB on the p53 IRES RNA. Studies on mutations that occur naturally in the 5' untranslated region (5' UTR) in p53 mRNA were lacking. We have investigated a naturally occurring C-to-T single-nucleotide polymorphism (SNP) first reported in human melanoma tumors. This SNP is at position 119 in the 5' UTR of p53 mRNA and we demonstrate that it has consequences on the translational control of p53. Introduction of this SNP has led to decrease in cap-independent translation from p53 5' UTR in bicistronic reporter assay. Further, the effects of this SNP on cap-independent translation have been studied in the context of p53 cDNA as well. Interestingly, the 5' UTR with this SNP has shown reduced binding to PTB that can be corroborated to its weaker IRES activity. Previously, it has been shown that G2-M checkpoint, DNA-damaging stress and oncogenic insult favor IRES-mediated translation. Under similar conditions, we demonstrate that this SNP interferes with the enhancement of the IRES activity of the 5' UTR. Taken together, the results demonstrate for the first time that SNP in the 5' UTR of the p53 mRNA might have a role in translational control of this critical tumor-suppressor gene.

Rab9A is required for delivery of cargo from recycling endosomes to melanosomes

Melanosomes are a type of lysosome-related organelle that is commonly defective in Hermansky-Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC-1, -2, -3, or AP-1, -3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A-depletion phenotype resembles Rab38/ 32-inactivated or BLOC-3-deficient melanocytes, suggesting that Rab9A works in line with BLOC-3 and Rab38/ 32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/ 32 or its effector VARP, or BLOC-3-deficiency in melanocytes decreased the length of STX13-positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co-regulatory GTPases control STX13-mediated cargo delivery to maturing melanosomes.