Håkan Hedman

LRIG1 restricts growth factor signaling by enhancing receptor ubiquitylation and degradation

Kekkon proteins negatively regulate the epidermal growth factor receptor (EGFR) during oogenesis in Drosophila. Their structural relative in mammals, LRIG1, is a transmembrane protein whose inactivation in rodents promotes skin hyperplasia, suggesting involvement in EGFR regulation. We report upregulation of LRIG1 transcript and protein upon EGF stimulation, and physical association of the encoded protein with the four EGFR orthologs of mammals. Upregulation of LRIG1 is followed by enhanced ubiquitylation and degradation of EGFR. The underlying mechanism involves recruitment of c‐Cbl, an E3 ubiquitin ligase that simultaneously ubiquitylates EGFR and LRIG1 and sorts them for degradation. We conclude that LRIG1 evolved in mammals as a feedback negative attenuator of signaling by receptor tyrosine kinases.

Epidermal growth factor receptor family (EGFR, ErbB2–4) in gliomas and meningiomas

Overexpression of epidermal growth factor receptor (EGFR, ErbB1) correlates with enhanced malignant potential of many human tumor types including glioblastoma multiforme. The significance of EGFR expression in meningiomas is, however, unclear. Reports regarding the other EGFR family members, ErbB2–4, in brain tumors are sparse. In this study, the expression of the EGFR family members was analyzed in relation to various parameters for the clinical importance of these receptors in 44 gliomas and 26 meningiomas. In gliomas, quantitative real-time reverse transcription (RT)-PCR revealed the highest EGFR mRNA expression in high-grade gliomas, while ErbB2 and ErbB3 mRNA were detected only in a few high-grade gliomas. In contrast, ErbB4 expression was most pronounced in low-grade gliomas. Immunohistochemistry showed significantly higher EGFR protein expression in high-grade gliomas compared to low-grade gliomas (P=0.004). ErbB2 protein expression was mainly seen in high-grade gliomas. ErbB3 protein expression was low in all gliomas analyzed. ErbB4 protein expression was significantly higher in low-grade gliomas than in high-grade gliomas (P=0.007). In meningiomas, quantitative real-time RT-PCR revealed expression of EGFR, ErbB2, and ErbB4 mRNA in the majority of the tumors. ErbB3 was detected in only one of the meningiomas analyzed. Immunohistochemistry demonstrated high ErbB2 protein expression in meningiomas. An intriguing observation in astrocytomas and oligodendrogliomas grade II, was a significantly decreased overall survival for patients with high EGFR protein expression (P=0.04). The high ErbB4 expression in low-grade compared to high-grade gliomas might suggest that ErbB4 acts as a suppressor of malignant transformation in brain tumors, which is in line with previous studies in other tumor types.

LRIG1 and epidermal growth factor receptor in renal cell carcinoma: a quantitative RT--PCR and immunohistochemical analysis.

In all, 31 renal cell carcinomas (RCCs) were examined for expression of the potential tumour suppressor LRIG1 (formerly Lig-1) and the epidermal growth factor receptor (EGFR). Eight matched samples of uninvolved kidney cortex were also evaluated. Gene expression was examined by quantitative real-time RT–PCR. In the eight matched sample pairs (uninvolved kidney cortex and tumour), protein expression was examined by immunohistochemistry. Conventional (clear cell) tumours showed an expected upregulation of EGFR. LRIG1 expression was generally downregulated in conventional and papillary RCC but not in chromophobic RCC. The ratio between EGFR and LRIG1 was more than 2.5-fold higher in the eight tumours compared with matched uninvolved kidney cortex and was at least two-fold higher than the mean normal ratio in 21 of 31 samples analysed. The observed downregulation of LRIG1 and increased EGFR/LRIG1 ratios are consistent with LRIG1 being a suppressor of oncogenesis in RCC by counteracting the tumour-promoting properties of EGFR. Further studies are justified to elucidate the explicit role of LRIG1 in the oncogenesis of RCC.

LRIG1 and squamous epithelial uterine cervical cancer: correlation to prognosis, other tumor markers, sex steroid hormones, and smoking

The aim is to evaluate LRIG1 as a prognosis predictor and correlations to cofactors in squamous cell cervical cancer. LRIG1 expression was studied in 128 cervical carcinomas and was compared with expression of nine other tumor markers. Smoking history was registered and pretreatment serum estradiol and progesterone levels were evaluated in 79 women. At clinical stage IB, 58% of the tumors showed LRIG1 expression, but there was a decline by increasing stage (33% in stage IV). Ninety percent of women with stage IB cancer and LRIG1 positivity survived, as compared to 64% without expression (P = 0.02). LRIG1 expression did not predict prognosis in advanced stages, but in stage IIA there was a marked relative difference, with 75% survival in tumors expressing LRIG1, as compared to 43% in those without. No correlation was found between LRIG1 and the other nine tumor markers studied. A high serum progesterone and smoking correlated to absent LRIG1 expression. We conclude that LRIG1 appears to be a significant prognosis predictor in early-stage cervical cancer, independent of the other tumor markers that were studied. Diminished expression in advanced stages and the inverse correlation to serum progesterone and smoking indicates that LRIG1 is a tumor suppressor in cervix.

Perinuclear leucine-rich repeats and immunoglobulin-like domain proteins (LRIG1-3) as prognostic indicators in astrocytic tumors

We have previously characterized three human leucine-rich repeats and immunoglobulin-like domains (LRIG) genes and proteins, named LRIG1-3 and proposed that they may act as suppressors of tumor growth. The LRIG1 transmembrane protein antagonizes the activity of epidermal growth factor receptor family receptor tyrosine kinases. In this study, we evaluated the mRNA expression level of LRIG1-3 in human glioma cell lines and control-matched glioma tissues, characterized the sub-cellular localization of an LRIG3–GFP fusion protein, and analyzed the relationship between sub-cellular localization of LRIG1-3 and clinical parameters in 404 astrocytic tumors by immunohistochemistry. LRIG1-3 mRNA was detected in all human glioma cell lines and matched glioma samples, with large differences in the expression levels. Ectopically expressed LRIG3–GFP localized to perinuclear and cytoplasmic compartments, and to the cell surface of transfected glioma cells. Perinuclear staining of LRIG1-3 was associated with low WHO grade and better survival of the patients. Perinuclear staining of LRIG3 was associated with a lower proliferation index and was in addition to tumor grade, an independent prognostic factor. Furthermore, within the groups of grade III and grade IV tumors, perinuclear staining of LRIG3 significantly correlated with better survival. These results indicate that expression and sub-cellular localization of LRIG1-3 might be of importance in the pathogenesis and prognosis of astrocytic tumors.

Cytoplasmic LRIG2 expression is associated with poor oligodendroglioma patient survival

The three leucine‐rich repeats and immunoglobulin‐like domains (LRIG) genes encode integral membrane proteins. Of these, LRIG1 negatively regulates growth factor signaling and is implicated as a tumor suppressor in certain malignancies. In astrocytic tumors, the subcellular distribution of LRIG proteins is associated with specific clinicopathological features and patient survival. The role of LRIG proteins in oligodendroglioma has not previously been studied. Here we used immunohistochemistry to analyze the expression of the LRIG proteins in 63 oligodendroglial tumors, and evaluated possible associations between LRIG protein expression and clinicopathological parameters. Notably, cytoplasmic LRIG2 expression was found to be an independent prognostic factor associated with poor oligodendroglioma patient survival. This is the first report of an LRIG protein showing a negative effect on survival, suggesting that LRIG2 might have a function different from that of LRIG1, and possibly contributing to the etiology of oligodendroglioma.

Dysregulated secretoglobin expression in human lung cancers

Lipophilins A, B, C, mammaglobin, and uteroglobin are members of the secretoglobin family of small, secreted, proteins. The functions of these proteins are not well understood but uteroglobin has been implicated in the development of cancers. Uteroglobin is known to be highly expressed in normal lung and down-regulated in lung cancers but expression of the other secretoglobins in normal lung and lung neoplasms have not been investigated. Therefore, we developed quantitative real-time reverse transcription (RT-) PCR assays for the different secretoglobins and evaluated their expression in normal and neoplastic lung tissues. The secretoglobin transcript levels were quantitated by real-time RT-PCR in samples from three normal lungs, 24 lung tumors including six small cell lung carcinomas, seven adenocarcinomas, and five squamous cell carcinomas, and in cell lines from three small cell lung carcinomas and one mesothelioma. Uteroglobin was confirmed to be abundantly expressed in normal lung and the different lung tumors showed down-regulated uteroglobin expression. Of the other secretoglobins, only lipophilin C was detected in normal lung, albeit at low levels. The lung tumors, however, frequently showed neo- or up-regulation of lipophilins A, B, C, and mammaglobin. The results constitute the first quantitative evaluation of secretoglobin expression in normal and neoplastic human lung tissues and demonstrate dysregulation in various human lung cancers. These findings could have important biological and diagnostic implications.

LRIG1 and the liar paradox in prostate cancer: a study of the expression and clinical significance of LRIG1 in prostate cancer

The course of prostate cancer varies greatly, and additional prognostic markers are needed. Leucine‐rich repeats and immunoglobulin‐like domains protein 1 (LRIG1) is an endogenous inhibitor of growth factor signaling and a proposed tumor suppressor. Publicly available gene expression datasets indicate that LRIG1 may be overexpressed in prostate cancer. In our study, the expression of LRIG1 protein in prostate cancer was evaluated for the first time. Immunohistochemistry was performed on tissue microarrays from two different patient series: 355 Swedish patients diagnosed by transurethral resection and 293 American patients who underwent radical prostatectomy. In the Swedish series, high expression of LRIG1 correlated with Gleason score, T‐stage, tumor cell proliferation, vascular density and epidermal growth factor receptor (EGFR) phosphorylation. Among the 256 Swedish patients, followed by watchful waiting, high LRIG1 expression was significantly associated with short overall and prostate cancer‐specific survival. In contrast, in the US series, high LRIG1 expression was significantly associated with long overall survival. In vitro cell experiments showed that LRIG1 was induced by androgen stimulation, and its expression inhibited prostate cancer cell proliferation. Thus, LRIG1 expression was an independent marker for poor survival in the untreated patient series, perhaps as a secondary marker of androgen receptor and/or EGFR activation. On the contrary, LRIG1 was a marker for good prognosis after prostatectomy, which might be due to its growth inhibiting properties. We propose that LRIG1 is an important determinant of prostate cancer growth, and the implications of its expression on patient outcome depend on the clinical and biological circumstances.

Paracrine regulation of growth factor signaling by shed leucine-rich repeats and immunoglobulin-like domains 1

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a recently discovered negative regulator of growth factor signaling. The LRIG1 integral membrane protein has been demonstrated to regulate various oncogenic receptor tyrosine kinases, including epidermal growth factor (EGF) receptor (EGFR), by cell-autonomous mechanisms. Here, we investigated whether LRIG1 ectodomains were shed, and if LRIG1 could regulate cell proliferation and EGF signaling in a paracrine manner. Cells constitutively shed LRIG1 ectodomains in vitro, and shedding was modulated by known regulators of metalloproteases, including the ADAM17 specific inhibitor TAPI-2. Furthermore, shedding was enhanced by ectopic expression of Adam17. LRIG1 ectodomains appeared to be shed in vivo, as well, as demonstrated by immunoblotting of mouse and human tissue lysates. Ectopic expression of LRIG1 in lymphocytes suppressed EGF signaling in co-cultured fibroblastoid cells, demonstrating that shed LRIG1 ectodomains can function in a paracrine fashion. Purified LRIG1 ectodomains suppressed EGF signaling without any apparent downregulation of EGFR levels. Taken together, the results show that the LRIG1 ectodomain can be proteolytically shed and can function as a non-cell-autonomous regulator of growth factor signaling. Thus, LRIG1 or its ectodomain could have therapeutic potential in the treatment of growth factor receptor-dependent cancers.

The soluble form of the tumor suppressor Lrig1 potently inhibits in vivo glioma growth irrespective of EGF receptor status

Background Deregulated growth factor signaling is a major driving force in the initiation and progression of glioblastoma. The tumor suppressor and stem cell marker Lrig1 is a negative regulator of the epidermal growth factor receptor (EGFR) family. Here, we addressed the therapeutic potential of the soluble form of Lrig1 (sLrig1) in glioblastoma treatment and the mechanism of sLrig1-induced growth inhibition. Methods With use of encapsulated cells, recombinant sLrig1 was locally delivered in orthotopic glioblastoma xenografts generated from freshly isolated patient tumors. Tumor growth and mouse survival were evaluated. The efficacy of sLrig1 and the affected downstream signaling was studied in vitro and in vivo in glioma cells displaying variable expression of wild-type and/or a constitutively active EGFR mutant (EGFRvIII). Results Continuous interstitial delivery of sLrig1 in genetically diverse patient-derived glioma xenografts led to strong tumor growth inhibition. Glioma cell proliferation in vitro and tumor growth in vivo were potently inhibited by sLrig1, irrespective of EGFR expression levels. Of importance, tumor growth was also suppressed in EGFRvIII-driven glioma. sLrig1 induced cell cycle arrest without changing total receptor level or phosphorylation. Affected downstream effectors included MAP kinase but not AKT signaling. Of importance, local delivery of sLrig1 into established tumors led to a 32% survival advantage in treated mice. Conclusions To our knowledge, this is the first report demonstrating that sLrig1 is a potent inhibitor of glioblastoma growth in clinically relevant experimental glioma models and that this effect is largely independent of EGFR status. The potent anti-tumor effect of sLrig1, in combination with cell encapsulation technology for in situ delivery, holds promise for future treatment of glioblastoma.