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Investigations of Leucine-rich repeats and immunoglobulin-like domain-proteins 1 and 2 (LRIG1 and LRIG2) and their genes in cancer
Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The mammalian leucine-rich repeats and immunoglobulin-like domains (LRIG) gene family consists of three different members, LRIG1, LRIG2, and LRIG3. These genes are expressed in all human and mouse tissues analyzed to date. All LRIG proteins share similar and evolutionary conserved structural domains including a leucine-rich repeat domain, three immunoglobulin-like domains, a transmembrane domain, and a cytosolic tail. Since the discovery of this family, around 20 years ago, various research groups have shown the importance of this family in cancer biology and prognosis. The aim of this thesis was to further investigate the role of LRIG1 and LRIG2 in cancer.

To investigate the roles of LRIG1 and LRIG2 in physiology and gliomagenesis, we generated Lrig1- and Lrig2-deficient mice and induced platelet-derived growth factor B (PDGFB)-driven gliomagenesis. We studied the effects of Lrig2 ablation on mouse development and survival and investigated if the ablation of Lrig1 or Lrig2 affects the incidence or malignancy of induced gliomas. We also investigated if Lrig2 ablation affects Pdgfr signaling in mouse embryonic fibroblasts (MEFs). Additionally, we analyzed the effects of ectopic LRIG1 expression in human primary glioblastoma cell lines TB101 and TB107, in vivo and in vitro. We reported no macroscopic anatomical defect but reduced growth and increased spontaneous mortality rate in Lrig2-deficient mice. However, the Lrig2-deficient mice were protected against the induced gliomagenesis. Lrig2-deficient MEFs showed faster kinetics of induction of immediate-early genes in response to PDGFB stimulation, whereas the phosphorylations of Pdgfra, Pdgfrb, Erk1/2, and Akt1 appeared unaltered. Lrig1-heterozygote mice showed a higher incidence of high-grade tumors (grade IV) compared to wildtype mice, demonstrating a haploinsufficient function of Lrig1. LRIG1 overexpression suppressed TB107 cell invasion in vivo and in vitro, which was partially mediated through the suppression of the MET receptor tyrosine kinase.

To identify LRIG1-interacting proteins, we used the yeast-two hybrid system and data-mined the Bio-Plex network of high throughput protein-protein interaction database. To study the function of interactors, we used a triple co-transfection system to overexpress LRIG1 and PDGFRA and downregulate endogenous levels of interactors by short hairpin RNAs (shRNAs), simultaneously. This analysis demonstrated that CNPY3, CNPY4, GAL3ST1, GML, HLA-DRA, LRIG2, LRIG3, LRRC40, PON2, RAB4A, and ZBTB16 were important for the PDGFRA-downregulating function of LRIG1.

To investigate the clinical significance of LRIG1 copy number alterations (CNAs) in breast cancer, we used droplet digital PCR (ddPCR) to analyze 423 breast cancer tumors. We found that LRIG1 CNAs were significantly different in steroid-receptor-positive vs steroid-receptor-negative tumors and in ERBB2-amplified vs ERBB2-non-amplified tumors. In the whole cohort, patients with LRIG1 loss or gain had a worse metastasis-free survival than patients with normal LRIG1 copy numbers, however, among the early-stage breast cancer subgroup, this difference was not significant. 

In summary, Lrig1 behaved like a haploinsufficient tumor suppressor gene in malignant glioma, whereas Lrig2 appeared to promote malignant glioma. Our functional analysis of LRIG1 interactome uncovered several unanticipated and novel proteins that might be important for the regulation of receptor tyrosine kinases by LRIG1. LRIG1 CNAs predicted metastasis-free survival time in breast cancer. Hopefully, our findings might lead to a better understanding of the regulation of growth factor signaling and its importance in cancer biology and prognosis.

 

Place, publisher, year, edition, pages
Umeå: Umeå university , 2018. , p. 79
Series
Umeå University Medical Dissertations, New Series No 1952, ISSN 0346-6612
Keywords [en]
LRIG1, LRIG2, PDGFR, glioma, interactome, breast cancer, prognosis
National Category
Cancer and Oncology
Research subject
Oncology
Identifiers
URN: urn:nbn:se:umu:diva-147388ISBN: 978-91-7601-895-8 (print)OAI: oai:DiVA.org:umu-147388DiVA, id: diva2:1203163
Public defence
2018-05-31, Betula, umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2018-05-09 Created: 2018-05-02 Last updated: 2018-06-09Bibliographically approved
List of papers
1. Lrig2-deficient mice are protected against PDGFB-induced glioma
Open this publication in new window or tab >>Lrig2-deficient mice are protected against PDGFB-induced glioma
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 9, p. e73635-Article in journal (Other academic) Published
Abstract [en]

Background: The leucine-rich repeats and immunoglobulin-like domains (LRIG) proteins constitute an integral membrane protein family that has three members: LRIG1, LRIG2, and LRIG3. LRIG1 negatively regulates growth factor signaling, but little is known regarding the functions of LRIG2 and LRIG3. In oligodendroglial brain tumors, high expression of LRIG2 correlates with poor patient survival. Lrig1 and Lrig3 knockout mice are viable, but there have been no reports on Lrig2-deficient mice to date. Methodology/Principal Findings: Lrig2-deficient mice were generated by the ablation of Lrig2 exon 12 (Lrig2E12). The Lrig2E12-/- mice showed a transiently reduced growth rate and an increased spontaneous mortality rate; 20-25% of these mice died before 130 days of age, with the majority of the deaths occurring before 50 days. Ntv-a transgenic mice with different Lrig2 genotypes were transduced by intracranial injection with platelet-derived growth factor (PDGF) B-encoding replication-competent avian retrovirus (RCAS)-producing DF-1 cells. All injected Lrig2E12+/+ mice developed Lrig2 expressing oligodendroglial brain tumors of lower grade (82%) or glioblastoma-like tumors of higher grade (18%). Lrig2E12-/- mice, in contrast, only developed lower grade tumors (77%) or had no detectable tumors (23%). Lrig2E12-/- mouse embryonic fibroblasts (MEF) showed altered induction-kinetics of immediate-early genes Fos and Egr2 in response to PDGF-BB stimulation. However, Lrig2E12-/- MEFs showed no changes in Pdgfr alpha or Pdgfr beta levels or in levels of PDGF-BB-induced phosphorylation of Pdgfr alpha, Pdgfr beta, Akt, or extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Overexpression of LRIG1, but not of LRIG2, downregulated PDGFR alpha levels in HEK-293T cells. Conclusions: The phenotype of Lrig2E12-/- mice showed that Lrig2 was a promoter of PDGFB-induced glioma, and Lrig2 appeared to have important molecular and developmental functions that were distinct from those of Lrig1 and Lrig3.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-71044 (URN)10.1371/journal.pone.0073635 (DOI)000324515600112 ()
Note

Included in thesis in manuscript form

Available from: 2013-05-17 Created: 2013-05-17 Last updated: 2018-06-08Bibliographically approved
2. Lrig1 is a haploinsufficient tumor suppressor gene in malignant glioma
Open this publication in new window or tab >>Lrig1 is a haploinsufficient tumor suppressor gene in malignant glioma
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2018 (English)In: Oncogenesis, E-ISSN 2157-9024, Vol. 7, article id 13Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-147385 (URN)10.1038/s41389-017-0012-8 (DOI)000429469500001 ()29391393 (PubMedID)
Available from: 2018-05-02 Created: 2018-05-02 Last updated: 2018-06-09Bibliographically approved
3. A protein interaction network centered on leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) regulates growth factor receptors
Open this publication in new window or tab >>A protein interaction network centered on leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) regulates growth factor receptors
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2018 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, no 9, p. 3421-3435Article in journal (Refereed) Published
Abstract [en]

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a tumor suppressor and a negative regulator of several receptor tyrosine kinases. The molecular mechanisms by which LRIG1 mediates its tumor suppressor effects and regulates receptor tyrosine kinases remain incompletely understood. Here, we performed a yeast two-hybrid screen to identify novel LRIG1-interacting proteins and mined data from the BioPlex (biophysical interactions of ORFeome-based complexes) protein interaction data repository. The putative LRIG1 interactors identified in the screen were functionally evaluated using a triple co-transfection system in which HEK293 cells were co-transfected with platelet-derived growth factor receptor α, LRIG1, and shRNAs against the identified LRIG1 interactors. The effects of the shRNAs on the ability of LRIG1 to down-regulate platelet-derived growth factor receptor α expression were evaluated. On the basis of these results, we present an LRIG1 protein interaction network with many newly identified components. The network contains the apparently functionally important LRIG1-interacting proteins RAB4A, PON2, GAL3ST1, ZBTB16, LRIG2, CNPY3, HLA-DRA, GML, CNPY4, LRRC40, and LRIG3, together with GLRX3, PTPRK, and other proteins. In silico analyses of The Cancer Genome Atlas data sets revealed consistent correlations between the expression of the transcripts encoding LRIG1 and its interactors ZBTB16 and PTPRK and inverse correlations between the transcripts encoding LRIG1 and GLRX3. We further studied the LRIG1 function–promoting paraoxonase PON2 and found that it co-localized with LRIG1 in LRIG1-transfected cells. The proposed LRIG1 protein interaction network will provide leads for future studies aiming to understand the molecular functions of LRIG1 and the regulation of growth factor signaling.

Place, publisher, year, edition, pages
The American Society for Biochemistry and Molecular Biology, 2018
Keywords
LRIG1, PDGFRA, PON2, PTPRK, ZBTB16, platelet-derived growth factor-C (PDGF-C), protein expression, protein-protein interaction, receptor tyrosine kinase, yeast two-hybrid
National Category
Basic Medicine
Identifiers
urn:nbn:se:umu:diva-147386 (URN)10.1074/jbc.M117.807487 (DOI)000426562800032 ()29317492 (PubMedID)
Available from: 2018-05-02 Created: 2018-05-02 Last updated: 2018-06-09Bibliographically approved
4. LRIG1 gene copy number analysis by ddPCR and correlation to clinical factors in breast cancecr
Open this publication in new window or tab >>LRIG1 gene copy number analysis by ddPCR and correlation to clinical factors in breast cancecr
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(English)Manuscript (preprint) (Other academic)
Keywords
LRIG1, prognosis, breast cancer
National Category
Clinical Medicine
Identifiers
urn:nbn:se:umu:diva-147387 (URN)
Available from: 2018-05-02 Created: 2018-05-02 Last updated: 2018-06-09

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