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Lung cancer is the leading cause of cancer-related death worldwide as well as in Sweden. Non-small cell lung cancer (NSCLC) is the predominant form, which is largely subdivided into adenocarcinomas and squamous cell carcinomas. A small minority of NSCLC cases that present with localized small tumors are curable with surgery alone, but adjuvant chemotherapy is recommended in most cases that are treated with surgery, even though it only increases the chance of cure by less than 5%. Therefore, additional biomarkers are needed to guide the clinical decision making in early-stage disease.

The leucine-rich repeats and immunoglobulin-like (LRIG) family of proteins consists of three paralogous transmembrane proteins that are involved in the regulation of growth factor signaling. Of these proteins, LRIG1 is the most studied and it interacts with a wide variety of growth factor receptors and related proteins, including the epidermal growth factor (EGFR) and several others. High levels of LRIG1 have been associated with better survival in a multitude of malignant diseases, including (but not limited to) breast cancer, bladder cancer, cervical cancer, glioma and melanoma. The aim of this thesis was to investigate whether LRIG1 was a prognostic factor in NSCLC as well, and to further characterize the biological role of LRIG proteins in this disease.

To investigate the prognostic impact of LRIG proteins in NSCLC, we stained a tissue microarray (TMA) containing tumor samples from 347 surgically treated early-stage NSCLC patients for LRIG1, LRIG2 and LRIG3. LRIG1 high-expressing adenocarcinoma cases had a large and statistically significant survival benefit of 33 months compared to negative cases. Similarly, an in silico analysis of a large gene expression dataset from the Oncomine database showed that high LRIG1 mRNA expression was linked to better survival as well. Differences in survival persisted even when adjusting for known prognostic factors, meaning that LRIG1 was an independent positive prognostic marker for survival in NSCLC.

A yeast two-hybrid screen was performed to search for proteins interacting with a conserved cytosolic peptide shared between all three mammalian LRIG proteins. This screen yielded hits for the paralogous proteins LIM domain only protein 7 (LMO7) and LIM and calponin homology domains-containing protein 1 (LIMCH1). LRIG1 and LRIG3 were both found to physically interact with LMO7 and LIMCH1 as assessed through immunoprecipitation techniques on overexpressed proteins. For LMO7, this could also be confirmed at endogenous protein levels using the proximity ligation assay. The 347 samples in the previously mentioned TMA were stained for LMO7. In our survival analysis, we observed no significant survival differences when looking at LMO7 alone, but the survival benefit observed for high LRIG1 was found to be limited to the subgroup that also was negative for LMO7. This means that the observed physical interaction between LRIG1 and LMO7 appears to translate to differences in patient survival.

To investigate possible mechanisms underlying the observed association between high LRIG1 expression and a favorable patient survival, a panel of NSCLC cell lines was modified to overexpress LRIG1. Broadly, LRIG1 overexpression resulted in minor decreases in cellular proliferation rates and no effects on chemosensitivity or radiosensitivity. Looking across the panel of NSCLC cell lines, no clear pattern was observed regarding the effects of LRIG1 overexpression on cellular motility. However, LRIG1 overexpression significantly decreased the clonogenic potential in most cell lines. The only cell line in the panel, H1299, that formed hematogenous disseminated disease in immunodeficient mice was used to establish a mixed-population primary tumor of tagged LRIG1 overexpressing cells and control cells. The LRIG1 transduced cells were was found to be enriched in the injected primary tumor, but no significant changes in their relative abundance was observed between the metastatic sites and their corresponding primary tumors.

In summary, we found that LRIG1 was an independent positive prognostic factor in early-stage NSCLC. We identified LMO7 and LIMCH1 as interaction partners for LRIG proteins and showed that the interaction between LMO7 and LRIG1 had implications for the clinical outcome in NSCLC. Furthermore, our mechanistic studies on the effects of LRIG1 overexpression on NSCLC cells suggested that the survival benefit conferred by high LRIG1 expression may be due to differences in metastatic potential. Taken together, the findings in this thesis suggest an important biological role for LRIG proteins in NSCLC.

Lungcancer är den vanligaste enskilda orsaken till cancerrelaterad död såväl i Sverige som världen. Icke småcellig lungcancer är den dominerande formen, som indelas ytterligare i adenokarcinom och skivepitelcancer. En fjärdedel av patienterna med icke småcellig lungcancer som har lokalt växande små tumörer kan botas med enbart kirurgi ibland med tillägg av adjuvant cytostatikabehandling. Denna behandling har biverkningar och ökar chansen till bot med högst 5%. Fler biomarkörer behövs därför för att styra det kliniska beslutsfattandet vid de tidiga sjukdomsstadierna av icke småcellig lungcancer.

Proteinfamiljen LRIG består av tre liknande transmembranproteiner som samtliga är involverade i regleringen av tillväxtfaktorsignalering. LRIG1 är det mest studerade proteinet av dessa och interagerar med ett stort antal tillväxtfaktorreceptorer och andra relaterade proteiner. Höga nivåer av LRIG1 har kopplats till bättre överlevnad vid ett stort antal cancersjukdomar, inklusive bland annat bröstcancer, urinblåsecancer, livmoderhalscancer, melanom och primära hjärntumörer. Målet med detta forskningsprojekt var att utröna om detta gäller även för icke småcellig lungcancer, och att ytterligare karakterisera de biologiska funktionerna hos LRIG-proteiner vid denna sjukdom.

För att undersöka den kliniska betydelsen av LRIG-proteiner vid icke småcellig lungcancer immunfärgade vi en tissue microarray (TMA) innehållandes vävnadsprover från 347 kirurgiskt behandlade NSCLC-patienter i tidigt stadium, med antikroppar mot LRIG1, LRIG2 och LRIG3. Patienter med adenokarcinom och höga nivåer av LRIG1 befanns ha en stor och statistiskt signifikant överlevnadsfördel jämfört med negativa fall, och överlevde i medeltal 33 månader längre. En analys av genexpressionsdata i Oncomine-databasen visade även att högt genuttryck av LRIG1 var kopplat till liknande överlevnadsfördelar. Skillnaderna i överlevnad kvarstod även efter justering för kända kliniska prognosfaktorer, vilket innebär att LRIG1 är en oberoende positiv prognostisk markör för överlevnad vid NSCLC.

En jäst två hybrid-screening utfördes för att leta proteiner som interagerade med ett evolutionärt bevarat proteinfragment som delas mellan alla LRIG-proteiner. Screeningen resulterade i träffar för de två närbesläktade proteinerna LMO7 och LIMCH1. LRIG1 och LRIG3 befanns båda interagera fysiskt med LMO7 och LIMCH1 vid immunprecipitation av överuttryckta proteiner. För LMO7 och LRIG1 respektive LRIG3 kunde vi med metoden proximity ligation assay påvisa att interaktionen sker även vid endogent uttryckta proteiner. Vidare kunde vi påvisa i en överlevnadsanalys att LMO7 ensamt inte har någon prognostisk effekt, men att överlevnadsfördelen vi observerade för höga nivåer av LRIG1 enbart gäller för patienter som samtidigt inte har förekomst av LMO7 i tumörvävnad. Detta betyder att den påvisade interaktionen mellan LRIG1 och LMO7 även medför skillnader i överlevnad.

För att undersöka mekanismer bakom de påvisade skillnaderna i överlevnad, modifierade vi cellinjer etablerade från icke småcellig lungcancer till att överuttrycka LRIG1. I stort resulterade överuttryck av LRIG1 endast i mindre skillnader i celldelningstakt och inga nämnvärda effekter på cellernas känslighet för vare sig cytostatika eller strålning. Däremot ledde överuttryck av LRIG1 till signifikant minskad klonogenicitet i de flesta cellinjerna. Den enda cellinjen i panelen som visade sig ha förmågan att bilda hematogena mikrometastaser, H1299, användes för att etablera en mixad primärtumör där spårbara celler med överuttryck av LRIG1 blandats med spårbara kontrollceller. LRIG1 anrikades i den injicerade primärtumören enbart, men cellsammansättningen var oförändrad när metastaser jämfördes med sina respektive primärtumörer.

Sammanfattningsvis kom vi fram till att LRIG1 var en oberoende positiv prognostisk faktor i tidiga stadier av icke småcellig lungcancer. Vi identifierade LMO7 och LIMCH1 som nya interaktionspartners för LRIG-proteiner och visade att interaktionen mellan LMO7 och LRIG1 har direkt relevans för det kliniska utfallet i lungcancer. Vidare visar våra mekanistiska studier på överuttryck av LRIG1 i tumörceller att överlevnadsfördelen åtminstone delvis beror på skillnader i förmågan att metastasera. Sammantaget pekar fynden i denna avhandling mot att LRIG-proteiner kan vara av viktig betydelse vid icke småcellig lungcancer.

Objectives: The human leucine-rich repeats and immunoglobulin-like domains (LRIG) protein family comprises the integral membrane proteins LRIG1, LRIG2 and LRIG3. LRIG1 is frequently down-regulated in human cancer, and high levels of LRIG1 in tumor tissue are associated with favorable clinical outcomes in several tumor types including non-small cell lung cancer (NSCLC). Mechanistically, LRIG1 negatively regulates receptor tyrosine kinases and functions as a tumor suppressor. However, the details of the molecular mechanisms involved are poorly understood, and even less is known about the functions of LRIG2 and LRIG3. The aim of this study was to further elucidate the functions and molecular interactions of the LRIG proteins.

Materials and methods: A yeast two-hybrid screen was performed using a cytosolic LRIG3 peptide as bait. In transfected human cells, co-immunoprecipitation and co-localization experiments were performed. Proximity ligation assay was performed to investigate interactions between endogenously expressed proteins. Expression levels of LMO7 and LIMCH1 in normal and malignant lung tissue were investigated using qRT-PCR and through in silico analyses of public data sets. Finally, a clinical cohort comprising 355 surgically treated NSCLC cases was immunostained for LMO7.

Results: In the yeast two-hybrid screen, the two paralogous proteins LMO7 and LIMCH1 were identified as interaction partners to LRIG3. LMO7 and LIMCH1 co-localized and co-immunoprecipitated with both LRIG1 and LRIG3. Endogenously expressed LMO7 was in close proximity of both LRIG1 and LRIG3. LMO7 and LIMCH1 were highly expressed in normal lung tissue and down-regulated in malignant lung tissue. LMO7 immunoreactivity was shown to be a negative prognostic factor in LRIG1 positive tumors, predicting poor patient survival.

Conclusion: These findings suggest that LMO7 and LIMCH1 physically interact with LRIG proteins and that expression of LMO7 is of clinical importance in NSCLC.

Recently, a genome-wide association study showed that a single nucleotide polymorphism (SNP) —rs11706832—in intron 2 of the human LRIG1 (Leucine-rich repeats and immunoglobulin-like domains 1) gene is associated with susceptibility to glioma. However, the mechanism by which rs11706832 affects glioma risk remains unknown; additionally, it is unknown whether the expression levels of LRIG1 are a relevant determinant of gliomagenesis. Here, we investigated the role of Lrig1 in platelet-derived growth factor (PDGF)-induced experimental glioma in mice by introducing mono-allelic and bi-allelic deletions of Lrig1 followed by inducing gliomagenesis via intracranial retroviral transduction of PDGFB in neural progenitor cells. Lrig1 was expressed in PDGFB-induced gliomas in wild-type mice as assessed using in situ hybridization. Intriguingly, Lrig1-heterozygous mice developed higher grade gliomas than did wild-type mice (grade IV vs. grade II/III, p = 0.002). Reciprocally, the ectopic expression of LRIG1 in the TB107 high-grade human glioma (glioblastoma, grade IV) cell line decreased the invasion of orthotopic tumors in immunocompromised mice in vivo and reduced cell migration in vitro. Concomitantly, the activity of the receptor tyrosine kinase MET was downregulated, which partially explained the reduction in cell migration. In summary, Lrig1 is a haploinsufficient suppressor of PDGFB-driven glioma, possibly in part via negative regulation of MET-driven cell migration and invasion. Thus, for the first time, changes in physiological Lrig1 expression have been linked to gliomagenesis, whereby the SNP rs11706832 may affect glioma risk by regulating LRIG1 expression.

Background: The leucine-rich repeats and immunoglobulin-like domains (LRIG) family of transmembrane proteins are involved in the regulation of cellular signal transduction. LRIG1 is an endogenous inhibitor of receptor tyrosine kinases (RTKs) and an emerging tumor suppressor. In the lung epithelium, the expression of LRIG1 is downregulated by tobacco smoking, and further downregulated in lung squamous cell carcinoma.

Material and methods: The expression of LRIG proteins were analyzed in 347 cases of non-small cell lung cancer (NSCLC) by immunohistochemistry, and LRIG1 mRNA expression was evaluated in 807 lung cancer samples in silico in the Oncomine database. Potential associations between the expression data and the clinical parameters, including patient survival, were investigated.

Results: Expression of the LRIG1 protein was found to be an independent prognostic factor in NSCLC, whereas expression of LRIG2 or LRIG3 did not correlate with patient survival. The levels of LRIG1 mRNA also correlated with the survival of NSCLC patients.

Conclusion: These findings demonstrate that LRIG1 is an independent prognostic factor in patients with NSCLC that could be important in future decision-making algorithms for adjuvant lung cancer treatment.

BACKGROUND: Optimal treatment decisions for cancer patients require reliable prognostic and predictive information. However, this information is inadequate in many cases. Several recent studies suggest that the leucine-rich repeats and immunoglobulin-like domains (LRIG) genes, transcripts, and proteins have prognostic implications in various cancer types. MATERIAL AND METHODS: Relevant literature was identified on PubMed using the key words lrig1, lrig2, and lrig3. LRIG mRNA expression in cancer versus normal tissues was investigated using the Oncomine database. RESULTS: The three human LRIG genes, LRIG1, LRIG2, and LRIG3, encode single-pass transmembrane proteins. LRIG1 is a negative regulator of growth factor signaling that has been shown to function as a tumor suppressor in vitro and in vivo in mice. The functions of LRIG2 and LRIG3 are less well defined. LRIG gene and protein expression are commonly dysregulated in human cancer. In early stage breast cancer, LRIG1 copy number was recently shown to predict early and late relapse in addition to overall survival; in nasopharyngeal carcinoma, loss of LRIG1 is also associated with poor survival. LRIG gene and protein expression have prognostic value in breast cancer, uterine cervical cancer, head-and-neck cancer, glioma, non-small cell lung cancer, prostate cancer, and cutaneous squamous cell carcinoma. In general, expression of LRIG1 and LRIG3 is associated with good survival, whereas expression of LRIG2 is associated with poor survival. Additionally, LRIG1 regulates cellular sensitivity to anti-cancer drugs, which indicates a possible role as a predictive marker. CONCLUSIONS: LRIG gene statuses and mRNA and protein expression are clinically relevant prognostic indicators in several types of human cancer. We propose that LRIG analyses could become important when making informed and individualized clinical decisions regarding the management of cancer patients.

Anaplastic lymphoma kinase (ALK) is an important molecular target in neuroblastoma. Although tyrosine kinase inhibitors abrogating ALK activity are currently in clinical use for the treatment of ALK-positive (ALK(+)) disease, monotherapy with ALK tyrosine kinase inhibitors may not be an adequate solution for ALK(+) neuroblastoma patients. Increased expression of the gene encoding the transcription factor MYCN is common in neuroblastomas and correlates with poor prognosis. We found that the kinase ERK5 [also known as big mitogen-activated protein kinase (MAPK) 1 (BMK1)] is activated by ALK through a pathway mediated by phosphoinositide 3-kinase (PI3K), AKT, MAPK kinase kinase 3 (MEKK3), and MAPK kinase 5 (MEK5). ALK-induced transcription of MYCN and stimulation of cell proliferation required ERK5. Pharmacological or RNA interference-mediated inhibition of ERK5 suppressed the proliferation of neuroblastoma cells in culture and enhanced the antitumor efficacy of the ALK inhibitor crizotinib in both cells and xenograft models. Together, our results indicate that ERK5 mediates ALK-induced transcription of MYCN and proliferation of neuroblastoma, suggesting that targeting both ERK5 and ALK may be beneficial in neuroblastoma patients.

Background: The malignant brain tumour glioblastoma is a devastating disease that remains a therapeutic challenge.

Materials and Methods: Effects of combinations of the US Food and Drug Administation (FDA) approved proteasome inhibitor bortezomib and the histone deacetylase (HDAC) inhibitors vorinostat, valproic acid and sodium phenylbutyrate were studied on primary glioblastoma stem cell lines and conventional glioblastoma cell lines. Cell survival, proliferation and death were analyzed by fluorometric microculture cytotoxicity assay (FMCA), propidium iodide labeling and flow cytometry, and cell cloning through limiting dilution and live-cell bright-field microscopy.

Results: Bortezomib and the HDAC inhibitors showed synergistic cell killing at clinically relevant drug concentrations, while the conventional cell lines cultured in serum-containing medium were relatively resistant to the same treatments.

Conclusion: These findings of synergistic glioblastoma stem cell killing by bortezomib and three different FDA-approved HDAC inhibitors confirm and expand previous observations on co-operative effects between these classes of drugs.