Laura Hautala

The DNA damage signalling kinase ATM is aberrantly reduced or lost in BRCA1/BRCA2-deficient and ER/PR/ERBB2-triple-negative breast cancer.

The ataxia-telangiectasia-mutated (ATM) kinase is a key transducer of DNA damage signals within the genome maintenance machinery and a tumour suppressor whose germline mutations predispose to familial breast cancer. ATM signalling is constitutively activated in early stages of diverse types of human malignancies and cell culture models in response to oncogene-induced DNA damage providing a barrier against tumour progression. As BRCA1 and BRCA2 are also components of the genome maintenance network and their mutations predispose to breast cancer, we have examined the ATM expression in human breast carcinomas of BRCA1/2 mutation carriers, sporadic cases and familial non-BRCA1/2 patients. Our results show that ATM protein expression is aberrantly reduced more frequently among BRCA1 (33%; P=0.0003) and BRCA2 (30%; P=0.0009) tumours than in non-BRCA1/2 tumours (10.7%). Furthermore, the non-BRCA1/2 tumours with reduced ATM expression were more often estrogen receptor (ER) negative (P=0.0002), progesterone receptor (PR) negative (P=0.004) and were of higher grade (P=0.0004). In our series of 1013 non-BRCA1/2 cases, ATM was more commonly deficient (20%; P=0.0006) and p53 was overabundant (47%; P<0.0000000001) among the difficult-to-treat ER/PR/ERBB2-triple-negative subset of tumours compared with cases that expressed at least one of these receptors (10 and 16% of aberrant ATM and p53, respectively). We propose a model of ‘conditional haploinsufficiency’ for BRCA1/2 under conditions of enhanced DNA damage in precancerous lesions resulting in more robust activation and hence increased selection for inactivation or loss of ATM in tumours of BRCA1/2 mutation carriers, with implications for genomic instability and curability of diverse subsets of human breast cancer.

Glycodelin in reproductive endocrinology and hormone-related cancer

Glycodelin is an endocrine-regulated glycoprotein that has significant effects on immune cells, apoptosis, reproduction, cell adhesion, differentiation and cancer. In reproduction, glycodelin contributes to capacitation and immunoprotection of spermatozoa, and it modulates sperm-oocyte binding, acrosome reaction and implantation. In endocrine-related cancer, the differentiation inducing effects of glycodelin are accompanied by growth restriction of malignant cells, decreased expression of oncogenes, increased expression of tumour suppressor genes and morphological reversion of the malignant phenotype. This review features these properties and clinical connections, highlighting the role of glycosylation in biological actions.

Human chorionic gonadotropin and its free β-subunit stimulate trophoblast invasion independent of LH/hCG receptor.

Both paracrine and autocrine factors are involved in the regulation of trophoblast invasion. One of these factors is human chorionic gonadotropin (hCG), which stimulates trophoblast invasion. The stimulatory activity has especially been ascribed to a hyperglycosylated form of hCG (hCG-h) that is expressed in early pregnancy. We compared the stimulatory activities of different forms of hCG and its free β-subunit (hCGβ) on trophoblast invasion. hCG, hCG-h, hCGβ, and its hyperglycosylated form (hCGβ-h) stimulated the invasion of JEG-3 choriocarcinoma cells. The stimulatory effect of hCGβ was also confirmed with primary human trophoblasts. Down-regulation of the LH/hCG receptor by RNA-interference did not significantly reduce the effect of hCGβ and hCG on cell invasion. Increased invasion was associated with increased levels of MMP-2, MMP-9 and activity of uPA. Our findings suggest that hCG, hCGβ and their hyperglycosylated forms stimulate the invasion of trophoblast cells independent of the classical LH/hCG-receptor.

Glycodelin reduces breast cancer xenograft growth in vivo

Malignant growth is characterized by loss of cell differentiation, uncontrolled proliferation and resistance to apoptosis. Many tumor suppressor genes that protect cells against malignant transformation regulate cell differentiation. Here, we show for the first time that glycodelin, a differentiation‐related protein, reduces breast cancer tumor growth in vivo. We found that glycodelin cDNA‐transfected MCF‐7 breast cancer cells showed a differentiated phenotype and produced smaller tumors in mouse mammary fat pads compared with control‐transfected cells. Glycodelin‐induced differentiation was associated with reduced expression of oncogenes and increased expression of tumor suppressor genes. Our results suggest that glycodelin acts as a tumor suppressor in breast cancer. This may explain its reported association with a more favorable prognosis in some cancers.

The role of glycodelin in cell differentiation and tumor growth

Glycodelin is a lipocalin family glycoprotein expressed mainly in reproductive tissues. It is involved in cell recognition, and its relationship with epithelial differentiation is well established. Glycodelin actually appears to drive epithelial differentiation. The evidence comes from studies employing endometrial and breast cancer cell lines. First, transfection of glycodelin cDNA into glycodelin‐negative carcinoma cells results in reduced expression of oncogenes, increased expression of tumor suppressor genes, increased cell differentiation, and reduced carcinoma cell growth. Second, histone deacetylase inhibitors (HDACIs) induce glycodelin synthesis in endometrial cancer cells concomitantly with cell differentiation. This effect is blocked by specific down‐regulation of glycodelin by RNA interference, suggesting that the effects of HDACIs are mediated by glycodelin. We recently found that glycodelin not only reduces carcinoma cell growth in vitro, but glycodelin cDNA transfection to MCF‐7 breast carcinoma cells also reduces growth of these cells in vivo, demonstrated by xenograft tumor growth in mouse mammary fat pads. These results strongly suggest that glycodelin acts as a tumor suppressor in breast cancer. The findings are compatible with the observations that certain types of glycodelin‐expressing ovarian and breast cancers have a more favorable prognosis compared to glycodelin non‐expressing tumors. This research has therefore introduced a novel mechanism to control cancer cell growth. In this communication we review the differentiation‐related effects of glycodelin

Absence of TGF-β Receptor Activation by Highly Purified hCG Preparations

Recently, several LH/human chorionic gonadotropin (hCG) receptor-independent activities for hCG have been described, including activation of the TGF-β receptor (TGFβR) by hyperglycosylated hCG and stimulation of trophoblast invasion. Because the hCG concentrations used in these studies have been rather high, reflecting physiological hCG levels in pregnancy, even a minor contamination with growth factors, which act at very low concentrations, may be significant. Several commercial hCG preparations have been found to contain significant amounts of epidermal growth factor (EGF), which we also confirmed here. Furthermore, we found that some hCG preparations also contain significant amounts of TGF-β1. These hCG preparations were able to activate ERK1/2 in JEG-3 choriocarcinoma cells or TGFβR in mink lung epithelial cells transfected with a reporter gene for TGFβR activation. No such activation was found with highly purified hCG or its free β-subunit (hCGβ), irrespective of whether they were hyperglycosylated or not. Taken together, our results suggest that the growth factor contaminations in the hCG preparations can cause activation of TGFβR and, at least in JEG-3 cells, MAPK signaling. This highlights the importance to carefully control for potential contaminations and that highly purified hCG preparations have to be used for biological studies.

Interleukin-6 increases expression of serine protease inhibitor Kazal type 1 through STAT3 in colorectal adenocarcinoma.

Inflammation promotes colorectal cancer (CRC) tumorigenesis, but the underlying molecular mechanisms are still being uncovered. Proinflammatory cytokine interleukin‐6 (IL‐6) stimulates survival signaling in CRC; inflammatory signals also regulate production and activity of proteases and their inhibitors. Over‐expression of serine protease inhibitor Kazal type 1 (SPINK1) predicts an unfavorable outcome in colon cancer. The SPINK1 gene contains an IL‐6 responsive element, suggesting it could act as an acute phase reactant. We assessed the connection between IL‐6 and SPINK1, and the function and mechanism of this signaling. Our results show that Colo205 and HT‐29 cells express and secrete SPINK1, and both fibroblast‐derived and recombinant IL‐6 further increased the SPINK1 levels. Concurrently CRC cells augmented the IL‐6 production in fibroblasts. In CRC tissues cancer cells were positive for SPINK1, whereas IL‐6 was found in stromal cells. In Colo205 cells IL‐6 also stimulated the secretion of trypsin‐1 and ‐2, the key targets of SPINK1 protease inhibition, whereas in HT‐29 cells trypsin‐1 and ‐2 levels remained constantly low. Functionally, both IL‐6 and SPINK1 increased the motility of the CRC cells. Mechanistically, IL‐6 activated the canonical STAT3 pathway and inhibition of STAT3 phosphorylation decreased the levels of SPINK1, trypsin‐1 and ‐2. Taken together, our results indicate a novel link between inflammatory signals originating from the tumor microenvironment and increased SPINK1 levels. This finding has potential therapeutic implications for targeted therapy, as it confirms that SPINK1 acts as an acute phase reactant and that it participates in the paracrine crosstalk with the tumor microenvironment of colon cancer.

Abstract 601: Glycodelin expression in lung cancer and melanoma

Glycodelin (PAEP) is a lipocalin protein mainly expressed in well-differentiated epithelial cells in reproductive tissues. Previously, glycodelin has been shown to induce cell differentiation in endometrial and breast cancer cells. In breast cancer this was associated with reduced xenograft tumor growth. In ovarian and breast cancers glycodelin expression has been found to associate with a favorable prognosis as compared to glycodelin non-expressing tumors. Some preliminary studies suggest that glycodelin expression is increased also in lung cancer and melanoma. We studied the expression of glycodelin in these cancers using immunohistochemical staining of tissue arrays. Furthermore, the levels of mRNA were addressed utilizing in silico transcriptomics database (IST Online). Glycodelin transcripts were more abundant in lung adenocarcinoma and melanoma as compared to non-malignant lung or squamous cell carcinoma of the lung and skin tissues, respectively. While immunohistochemical staining also showed increased staining of glycodelin in lung adenocarcinoma as compared to squamous cell carcinoma of the lung, there was no clear difference in staining as compared to normal lung tissue. Weak staining of glycodelin was observed both in normal skin and melanoma tissues. However, some melanoma cases showed very strong staining. In conclusion, our data show that glycodelin is expressed both in melanoma and lung adenocarcinoma, and in some cases the expression is increased. However, the clinical relevance of these findings remains to be established. Citation Format: Laura Hautala, Hannu Koistinen. Glycodelin expression in lung cancer and melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 601. doi:10.1158/1538-7445.AM2015-601

Abstract 4066: Glycodelin abolishes PMA-induced migration of MCF-7 breast cancer cells

Glycodelin is a lipocalin protein mainly expressed in well-differentiated epithelial cells in reproductive tissues. Previously, glycodelin has been shown to induce cell differentiation in endometrial and breast cancer cells. We have used glycodelin-transfected MCF-7 breast cancer cells to study the glycodelin-induced differentiation. Glycodelin expression changed the cellular morphology towards normal, less malignant direction. In addition, glycodelin-producing cells formed significantly smaller tumors in xenograft mice. We have found that the effects of glycodelin are at least partly mediated by repressed PKCδ activity, rendering the cells resistant to activation of phorbol 12-myristate 13-acetate (PMA), a known activator of PKCδ. Here we continued these previous studies by addressing the effect of glycodelin on the invasion and migration of MCF-7 breast cancer cells, and studying the affected signaling route in more detail. Activation of signaling proteins was detected with an antibody array of 43 phospho kinases. Cell migration was studied using wound healing test. The cells were grown on chamber slides until they formed a monolayer and then a scratch was made using a pipet tip. After addition of PMA or DMSO control, the migration of the cells was monitored under microscope. In some of the control cells, PKCδ was down-regulated with RNAi prior the migration assay. For invasion through Matrigel basement membrane preparation, a Boyden chamber assay was used. The cells were grown with and without PMA and the amount of invasive cells was quantified. Phospho-kinase array did not reveal any widespread changes in the amount of phosphorylated signaling molecules between the glycodelin-producing and control cells irrespective of the PMA treatment. However, in addition to PKCδ some changes were found in the levels of phosphoproteins such as p38α and p27. In wound healing test PMA caused a two-fold increase in the migration of the control cells, but had no effect on the glycodelin-producing cells. When the expression of PKCδ was blocked in the control cells using siRNA, the addition of PMA did not increase the migration of the cells. PMA also increased the invasiveness of the cells, but that was detected in both cells clones regardless of glycodelin expression. Without PMA treatment both glycodelin-expressing and control cells behaved similarly in wound healing and invasion tests. In conclusion, glycodelin abolished PMA-induced migration in MCF-7 breast cancer cells, while it did not have any effect on the invasion of the cells. Our results suggest that the effect of glycodelin in migration is mediated by reduced activation of PKCδ. Citation Format: Laura C. Hautala, Riitta Koistinen, Hannu Koistinen. Glycodelin abolishes PMA-induced migration of MCF-7 breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4066. doi:10.1158/1538-7445.AM2014-4066

Repressed PKCδ activation in glycodelin-expressing cells mediates resistance to phorbol ester and TGFβ

Glycodelin is a glycoprotein mainly expressed in well-differentiated epithelial cells in reproductive tissues. In normal secretory endometrium, the expression of glycodelin is abundant and regulated by progesterone. In hormone-related cancers glycodelin expression is associated with well-differentiated tumors. We have previously found that glycodelin drives epithelial differentiation of HEC-1B endometrial adenocarcinoma cells, resulting in reduced tumor growth in a preclinical mouse model. Here we show that glycodelin-transfected HEC-1B cells have repressed protein kinase C delta (PKCδ) activation, likely due to downregulation of PDK1, and are resistant to phenotypic change and enhanced migration induced by phorbol 12-myristate 13-acetate (PMA). In control cells, which do not express glycodelin, the effects of PMA were abolished by using PKCδ and PDK1 inhibitors, and knockdown of PKCδ, MEK1 and 2, or ERK1 and 2 by siRNAs. Similarly, transforming growth factor β (TGFβ)-induced phenotypic change was only seen in control cells, not in glycodelin-producing cells, and it was mediated by PKCδ. Taken together, these results strongly suggest that PKCδ, via MAPK pathway, is involved in the glycodelin-driven cell differentiation rendering the cells resistant to stimulation by PMA and TGFβ.