| 1. |
- Holmlund, Camilla, 1969-
(författare)
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Identification and investigations of leucine-rich repeats and immunoglobulin-like domains protein 2 (LRIG2)
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Receptor tyrosine kinases (RTKs) constitute a family of proteins controlling cell growth and proliferation and whose activities are tightly controlled in normal cells. LRIG1 is a negative regulator of RTK signaling and is a proposed tumor suppressor. The aim of this thesis was to identify and study possible paralogs of LRIG1. By using the basic local alignment search tool and cDNA cloning, a human mRNA sequence with similarity to LRIG1 was identified and named LRIG2. By fluorescence in situ hybridization analysis, LRIG2 was found to reside on chromosome 1p13. The LRIG2 amino acid sequence was 47% identical to LRIG1, and the predicted protein domain organization was the same as that of LRIG1. Antibodies against LRIG2 were developed and the apparent molecular weight of the protein was determined to be 132 kDa by SDS-polyacrylamide gel electrophoresis and Western blot analysis. The sub-cellular localization was studied by cell surface biotinylation experiments and confocal fluorescence laser microscopy, which revealed that LRIG2 resided at the cell surface and in the cytoplasm. The expression patterns of LRIG2 mRNA, during development and in adult tissues, were evaluated using whole-mount in situ hybridization and quantitative real-time RT-PCR, respectively. In E10.5, E11.5 and E12.5 mouse embryos, the Lrig2 expression domains were both overlapping and unique as compared to the expression domains of Lrig1 and the third family member, Lrig3. In adult human tissues, the most prominent LRIG2 mRNA expression was found in skin, uterus and ovary. To study the developmental and physiological role of LRIG2, Lrig2 knock-out mice were generated. The knock-out mice were born at Mendelian frequencies without any apparent morphological abnormalities. However, Lrig2 knock-out mice showed reduced body weight between 5 days and 12-15 weeks of age, increased mortality, and impaired reproductive capacity. To study the role of LRIG2 as a prognostic factor in oligodendroglioma, LRIG2 expression was analyzed in 65 human oligodendrogliomas by immunohistochemistry. Cytoplasmic LRIG2 expression was an independent prognostic factor associated with poor oligodendroglioma patient survival. The possible functional role of LRIG2 in oligodendroglioma biology was further investigated using the RCAS/tv-a mouse model. Tumors resembling human oligodendroglioma were induced by intracranial injection of PDGFB carrying RCAS retroviruses into newborn Ntv-a mice. Lrig2 wild-type animals developed tumors at a higher frequency and of higher malignancy than the Lrig2 knock-out mice. This result supports the notion that LRIG2 promotes PDGF-induced oligodendroglioma genesis. A possible molecular mechanism was revealed as LRIG2 overexpression increased PDGFRa levels in transfected cells. In summary, we identified a new gene named LRIG2, showed that it is expressed in a variety of tissues during development and in adulthood, knocked it out and found that it was required for proper animal growth, health, and reproduction. We also found that Lrig2 expression promoted PDGF-induced oligodendroglioma genesis and was associated with poor oligodendroglioma patient survival, possibly via a PDGFRa stabilizing function.
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| 2. |
- Karlsson, Terese, 1979-
(författare)
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The expression and molecular functions of LRIG proteins in cancer and psoriasis
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The leucine-rich repeats and immunoglobulin-like domains (LRIG) family consists of three integral membrane proteins that are important in human cancer. LRIG1 is a negative regulator of growth factor signaling. Its expression is associated with longer survival in several cancer types, and the gene has been shown to function as a tumor suppressor. The roles of LRIG2 and LRIG3 are less well known. The aim of this thesis was to improve our understanding of the expression and function of the LRIG protein family in psoriasis and cancer.To investigate their expression in psoriasis, the mRNA levels and subcellular localization of the LRIG proteins were analyzed and compared between normal and psoriatic human skin. There were no differences in the LRIG mRNA levels between psoriatic and normal skin samples. However, the subcellular localization of all three LRIG proteins differed between psoriatic and normal skin.To study the physiological and molecular functions of Lrig2, we generated Lrig2E12-/- mice. These mice were viable and born at a Mendelian rate, but Lrig2E12-/- mice had an increased rate of spontaneous mortality and a transient reduction in growth rate compared to Lrig2 wild-type (wt) mice. In an orthotopic platelet-derived growth factor (PDGF)B-driven brain tumor mouse model, we studied the effect of Lrig2 on gliomagenesis. All Lrig2 wt mice developed tumors; 82% developed grade II/III tumors, and 18% developed grade IV tumors. Only 77% of the Lrig2E12-/- mice developed tumors, and they were all grade II/III tumors. Thus, Lrig2 increased the incidence and malignancy rates of PDGFB-driven gliomas. We then analyzed the effect of Lrig2 on Pdgf receptor (Pdgfr) signaling. Lrig2 had no effect on Pdgfr steady-state levels, the starvation-induced up-regulation of Pdgfrs, the phosphorylation of Pdgfrs, primary cilium formation or the PDGFBB-induced phosphorylation of Akt or Erk1/2. However, the kinetics of induction of the immediate-early genes Fos and Egr2 were altered, resulting in a more rapid induction in Lrig2E12-/- cells.We then analyzed the clinical and biological importance of LRIG1 in lung cancer. In a human lung cancer tissue micro-array (TMA), LRIG1 expression was found to be an independent positive prognostic factor for adenocarcinoma. To study the importance of Lrig1 regarding lung cancer development in vivo, we used an inducible EGFRL858R-driven mouse lung cancer model. The mice developed diffuse lung adenocarcinoma, and the tumor burden was greater in Lrig1-/- mice than in Lrig1+/+ mice (p = 0.025) at 60 days. The human lung cancer cell line H1975, with either normal or Tet-induced expression of LRIG1, was injected into the flanks of Balb/cA nude mice. Tumors formed by LRIG1-overexpressing cells were smaller than those formed by parental cells, further indicating that LRIG1 is important during lung tumor formation or growth. In vitro, LRIG1 suppressed the proliferation of H1975 cells and down-regulated the phosphorylation of MET and RET.To investigate the molecular functions of LRIG proteins further, we performed a yeast two-hybrid (YTH) screen using a peptide from the cytosolic tail of LRIG3 as bait. This screen identified LMO7 and LIMCH1 as prominent interaction partners for LRIG3. Proximity ligation assays showed that LMO7 interacted with all of the LRIG proteins at endogenous expression levels. LMO7 and LIMCH1 were expressed in all human tissues analyzed. Their expression was dramatically decreased in lung cancer compared to normal lung tissue. The expression of LMO7 was analyzed in a human lung cancer TMA. LMO7 was expressed in respiratory epithelial cells in normal lungs. However, LMO7 was only expressed in a quarter of the lung tumors. LMO7 expression was found to be an independent negative prognostic factor for lung cancer.In summary, we found that the LRIG proteins were redistributed in psoriatic skin. In a mouse glioma model, Lrig2 promoted oligodendroglioma genesis. LRIG1 was an independent positive prognostic factor in human lung cancer. Lrig1 ablation increased the tumor size in an EGFRL858R-driven lung cancer mouse model. LRIG1 expression decreased the tumor growth of human lung cancer cells in a xenograft mouse model. LMO7 interacted with all three LRIG proteins and was an independent negative prognostic factor in human lung cancer. These data demonstrate the importance of LRIG proteins in human disease.
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| 3. |
- Lindström, Annika, 1953-
(författare)
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Prognostic factors for squamous cell cervical cancer : tumor markers, hormones, smoking, and S-phase fraction
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cervical cancer is the second most common malignancy in women worldwide and one of the leading causes of cancer mortality globally. In patients with invasive cervical cancer prognostic factors are of value for the choice of treatment, monitoring of treatment and follow-up. The most important clinical prognostic factors are stage, tumor volume, parametrial infiltration, vascular invasion, lymph node metastases, and distant metastases. An improved estimation of the prognosis of cervical cancer is desirable, especially in early cancer stages.The aim of this research was to study possible associations between tumor markers, female sex steroids, smoking, S-phase fraction (SPF), and prognosis in invasive squamous cell cervical cancer (SCC). The study comprised 190 patients with SCC, stages IB-IV, admitted to the Department of Gynecologic Oncology at Norrland University Hospital in Umeå between September 1984 and October1990. Ten year mortality was estimated.In study I, of a total of 103 patients, it was found that increased tumor growth, measured by the DNA SPF, was associated with elevated serum progesterone and smoking in the premenopasual patients and with aneuploidy in the whole group.In study II, comprising 128 patients, survival length related to hormone levels and SPF was evaluated in women who died of cervical cancer. In both pre- and postmenopausal women, who died of cervical cancer, SPF at or above 12% was correlated with reduced survival. There was significant positive correlation between a low serum estradiol/progesterone ratio and short survival in those premenopausal women who died of cancer (p=0.02).In study III, ten-year follow-up results in 128 women were compared with the expression of ten relevant tumor markers, assessed by immunohistochemistry. The overall ten-year survival rate in patients with low COX-2 and high CD4+ expression was 76%, versus 53% in the remaining women. The survival rate with absent p53 and high COX-2 expression in the tumors was 42%, versus 71%, while the corresponding figure for the combination of high COX-2 intensity and expression of c-myc was 27%, versus 62%. None of the single markers correlated significantly with outcome in the final Cox regression analyses, while five combinations did.Study IV addressed possible associations between selected tumor markers and cofactors in SCC. Ten tumor markers were examined in 128 patients. Smoking habits and previous oral contraceptive use were recorded. Serum estradiol and progesterone levels were evaluated in 80 women. Highly significant associations were found between strong c-myc staining and increased progesterone, low EGFR staining and high serum estradiol, and absence of p53 staining and smoking. There was an association between absence of p53 and high serum progesterone.In study V, LRIG1 expression was studied in 128 patients and was compared with expression of nine other tumor markers, smoking history, hormone levels, and prognosis. LRIG1 appears to be a significant prognostic predictor in early stage SCC, independent of the other tumor markers that were studied. Diminished expression in advanced cancer stages and the inverse correlation to serum progesterone and smoking indicate that LRIG1 is a tumor suppressor in squamous cell cervical cancer.Conclusion: The results of these studies support a role of progesterone as a promoter of cervical cancer and indicate that smoking is associated with tumor progression. A combination of tumor markers might be of help in prognostic prediction. LRIG1 acts as a tumor suppressor. These findings might contribute towards greater understanding of prognostic prediction of squamous cell cervical cancer.
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