Anna Göthlin Eremo

Background: Blockade of IL1RAP (interleukin 1 receptor associated protein) was recently shown to reduce atherosclerosis in mice, but the effect on human vascular cells is largely unknown. Targeting the IL1RAP coreceptor represents a novel strategy to block the IL1RAP‐dependent cytokines IL (interleukin)‐1, IL‐33, and IL‐36. In the present study, we aimed to evaluate the role of novel antibodies targeting IL1RAP to reduce the effects of IL‐1β, IL‐33, or IL‐36γ in human vascular cells. Methods: Expression of IL1RAP was observed in human atherosclerotic plaques by immunohistochemistry and microarray and in endothelial cells by flow cytometry. Endothelial cells were cultured with IL‐1β, IL‐33, or IL‐36γ cytokines with or without IL1RAP antibodies and analyzed with Olink proteomics, ELISA, Western blot, and real‐time quantitative polymerase chain reaction. The functional effect of IL1RAP antibodies on endothelial cells were analyzed with adhesion and permeability assays. Results: Olink proteomics showed inhibition of the inflammatory proteins LIF (leukemia inhibitory factor), OPG (osteoprotegerin), CCL4 (C‐C motif chemokine ligand 4), and MCP‐3 (monocyte chemoattractant protein 3) by IL1RAP‐blockade in endothelial cells after IL‐1β stimulation. In addition, the IL1RAP antibodies inhibited IL‐1β, and IL‐33 induced IL‐6 and IL‐8 secretion. Secretion of MCP‐1 (monocyte chemoattractant protein 1) was induced by IL‐1β, IL‐33, and IL‐36γ, and subsequently was inhibited by IL1RAP antibodies. Similar effects were found on mRNA expression level. Endothelial expression of the adhesion markers ICAM1 , VCAM1, and SELE were significantly reduced by IL1RAP antibodies, and neutrophil adhesion to endothelial cells induced by IL‐1β and IL‐33 was reduced by IL1RAP blockade. In human atherosclerotic lesions, IL1RAP expression correlated with markers of inflammation like IL6 , IL8, and MCP1 . Conclusions: IL1RAP‐targeting antibodies can reduce the expression of inflammatory cytokines and markers of adhesion in endothelial cells, which may be of importance for future putative targeted treatments against cardiovascular disease.

Objective: Increased expression of the amino acid transporter solute Carrier Family 7 Member 5 (SLC7A5) has been observed in neoplastic cells during hypoxic conditions in vitro, indicating an adaptation for cell survival. To further explore this, we evaluated hypoxia-mimetic by CoCl2 as a model for hypoxia in breast cancer cell lines and the effect on SLC257A5 expression. Four different breast cancer cell lines (MCF7, T-47D, BT-474 and ZR-75-1) were exposed to 100 µM CoCl₂ for 48 h. Subsequently, cell viability, gene- and protein expression analyses were performed. Results: The gene expression of VEGF, a marker of hypoxia, was significantly elevated in all four cell lines compared to the control (p < 0.0001), indicating that CoCl₂ exposure generates a hypoxic response. Moreover, CoCl₂ exposure significantly upregulated SLC7A5 gene expression in T-47D (p < 0.001), BT-474 (p < 0.0001) and ZR-75-1 (p < 0.0001) cells, as compared to vehicle control. Immunofluorescence staining showed increased SLC7A5 protein expression in MCF7, T-47D and BT-474 cells compared to vehicle control. This report suggests that hypoxia-mimetic by CoCl₂ can be used as a simple model for inducing hypoxia in breast cancer cell lines and in fact influence SLC7A5 gene and protein expression in vitro.

Background Basal cell carcinoma (BCC) is the most common type of cancer in fair-skinned individuals worldwide. Altered metabolism is a hallmark of cancer, and a growing body of evidence has shown increased expression of the large neutral amino acid transporter (LAT) small subunit 1 in several types of cancers, including BCC. However, the mechanisms behind changed LAT1 expression in BCC are largely unknown. Objectives To describe the protein expression of LAT1 and its co-localisation with LAT2, and to examine LAT1 in association with BCC tumour biology characteristics such as cell proliferation, apoptosis, and hypoxia. Methods Formalin-fixed and paraffin-embedded tissue samples (n=14) from excised BCCs were stained with immunofluorescence and examined regarding protein-staining patterns. Results There was no correlation between expression of LAT1 and LAT2, and the co-localisation was low. The proliferation markers topoisomerase IIα and Ki-67 both showed a significantly higher expression in the BCC tissue than in the normal epidermis (p=0.0063 and p=0.010, respectively). The fraction of LAT1-expressing cells in the BCC was inversely correlated to the fraction of proliferative active tumour cells (p=0.0013). Cleaved caspase-3 was significantly increased in tumour areas with high LAT1 expression (p=0.016). Conclusions The findings of the present study show that LAT1 is not usually expressed by proliferating BCC cells. The morphological localisation suggests that tumour cells use LAT1 in adaption to environmental changes such as starvation and/or hypoxia. These findings could have implications for future development of LAT1-inhibitory BCC treatments.

The amino acid transporter named solute carrier family 7 member 5 (SLC7A5) is suggested to play a part in altered cell metabolism and proliferative signaling and has been reported to be overexpressed in various types of cancer, including breast cancer. Estrogen‑receptor‑positive (ER⁺) breast cancers constitute the most common type of breast malignancies and are often treated with anti‑estrogenic therapies. In this group of patients, endocrine resistance is a challenging problem that could lead to recurrent disease. To overcome this, additional prognostic biomarkers are needed. The present study aimed therefore to determine whether SLC7A5 may be considered as a possible prognostic marker in ER⁺ breast cancer and to investigate its relation with certain cancer‑related genes. We used a local breast cancer cohort (n=154) and immunohistochemistry to analyze the expression of SLC7A5 in association with clinicopathological characteristics and patient outcome. In addition, gene expression analysis was performed on 80 of these tumors. Furthermore, the METABRIC dataset was used for correlation analyses between expression of SLC7A5 and several genes related to breast cancer biology. The results demonstrated that overexpression of SLC7A5 was significantly associated with histopathological grade in patients with breast cancer, and that SLC7A5 mRNA expression was positively correlated with the expression of marker of proliferation Ki‑67 and hypoxia inducible factor 1 subunit alpha. Overexpression of SLC7A5 may therefore play a role in the biology of endocrinologically‑driven disease. However, when further assessing SLC7A5 using the METABRIC dataset, SLC7A5 mRNA expression level was more significantly increased in ER‑ subgroups compared with ER⁺ disease. All breast cancer subtypes included, SLC7A5 mRNA expression was correlated with a higher number of cancer‑related genes than in estrogen receptor positive tumors alone. The present study suggested that SLC7A5 expression may be of importance for breast cancer cell proliferation and survival. In order to further establish the biological and clinical role of SLC7A5 in breast cancer, further investigation using different breast cancer subgroups is required.

Breast cancer patients treated with tamoxifen may experience recurrence due to endocrine resistance, which highlights the need for additional predictive and prognostic biomarkers. The glyco-phosphoprotein osteopontin (OPN), encoded by the SPP1 gene, has previously shown to be associated with poor prognosis in breast cancer. However, studies on the predictive value of OPN are inconclusive. In the present study, we evaluated tissue SPP1 mRNA and OPN protein expression as markers of recurrence in estrogen receptor- positive (ER+) breast cancer tissue. Tamoxifen- treated patients with recurrence or non-recurrence were selected using a matched case-control design. SPP1 mRNA expression was analysed using qPCR (n = 100) and OPN protein by immunohistochemistry (n = 116) using different antibodies. Odds ratios were estimated with conditional logistic regression. The SPP1 expression increased the risk of recurrence with an odds ratio (OR) of 2.50 (95% confidence interval [CI]; 1.30–4.82), after adjustment for tumour grade, HER 2 status and other treatments to OR 3.62 (95% CI; 1.45–9.07). However, OPN protein expression was not associated with risk of recurrence or with SPP1-gene expression, suggesting SPP1 mRNA a stronger prognostic marker candidate compared to tumor tissue OPN protein.

Introduction The incidence of basal cell carcinoma (BCC) is increasing and the costs for care rising. Therefore the need of simplified and cost‐effective treatment choices is substantial. Aberrant signalling in several pathways, induced by UV‐radiation, is of importance in the development of BCC. Alterations in tumour metabolic activity are part of general carcinogenesis, however only partially recognized in skin cancer. Aim To study expression profiles in BCCs compared to individually matched non‐tumour skin, with focus on finding differences associated to tumour metabolism. Materials and methods Gene expression in biopsies from BCC (n =14) compared to biopsies from non‐ tumour gluteal skin was analysed with microarray (n = 4 + 4) and/or quantitative polymerase chain reaction (qPCR, n = 14 + 14). Protein expression and localisation was assessed using immunohistochemistry (IHC) in formalin fixed and paraffin embedded BCC. Results Microarray analysis revealed increased expression of the amino acid transporters SLC7A5, SLC7A7 and SLC7A8 as well as the tryptophan 2,3‐dioxygenase (TDO) 2 in BCC. Higher expression of SLC7A5 (P < 0.0001), SLC7A8 (P < 0.0001) and TDO2 (P = 0.0017), but not SLC7A7 (P = 0.50), wasconfirmed by qPCR and IHC demonstrated correlating tumour cell protein expression of SLC7A5 and SLC7A8. Protein expression of SLC7A7 was observed in stratum granulosum and TDO2 in immune cells. Conclusions This study highlights the upregulation of SLC7A5, SLC7A8 and TDO2 in BCC compared to non‐tumour skin. Our findings imply that amino acid transporters may be further explored as potential targets for future medical treatment.

Basal cell carcinoma is the most common type of cancer in fair-skinned individuals, and its incidence is rapidly increasing. The aim of the present study was to investigate the gene and protein expression of the mitochondrial solute carrier family 25 member 43 (SLC25A43) in basal cell carcinoma. SLC25A43 has previously been identified to be genetically altered and associated with cell proliferation in human epidermal growth factor receptor 2-positive breast cancer. However, the knowledge about SLC25A43 is limited, and its role in other cancers is unknown. The SLC25A43 gene and protein expression was analysed in 14 basal cell carcinomas and healthy skin samples from the same individuals by quantitative polymerase chain reaction and immunohistochemistry, respectively. The results demonstrated a significantly lower (≥50%) SLC25A43 gene expression in all carcinomas compared with that in healthy skin. In addition, SLC25A43 protein expression was absent in >90% of all visual fields in the basal cell carcinomas, and the H-score was significantly lower in tumours compared with the adjacent epidermis. These results demonstrate that SLC25A43 expression is altered at the gene and protein levels in basal cell carcinoma. The underlying mechanisms and the clinical relevance of these data must be elucidated in additional experimental and clinical studies.

The human epidermal growth factor receptor (HER) 4 is a relative of HER2 and has been associated to endocrine breast cancer and prediction of tamoxifen response. In addition to PI3K/Akt and MAPK pathway activation, ligand binding to HER4 triggers proteolytic cleavage and release of an intracellular receptor domain (4ICD) with signaling properties. The aim of the present study was to analyze HER4 protein expression and intracellular localization in breast cancer tissue from patients randomized to treatment with or without adjuvant tamoxifen. To investigate HER4 expression and localization in response to estradiol (E2) and 4-hydroxytamoxifen (4-OHT) exposure, we also performed in vitro studies. Cytoplasmic, nuclear and membrane expression of HER4 protein was evaluated by immunohistochemical staining in tumor tissue from 912 breast cancer patients. Three different breast epithelia cancer cell lines were exposed to E2 and 4-OHT and mRNA expression was analyzed using qPCR. Further, nuclear and cytoplasmic proteins were separated and analyzed with western blotting. We found an association between nuclear HER4 protein expression and ER-positivity (P=0.004). Furthermore, significant association was found between cytoplasmic HER4 and ER-negativity (P<0.0005), PgR-negativity (P20 mm (P=0.001) and HER2-negativity (P=0.008). However, no overall significance of HER4 on recurrence-free survival was found. After E2 exposure, HER4 mRNA and protein expression had decreased in two cell lines in vitro yet no changes in nuclear or cytoplasmic protein fractions were seen. In conclusion, nuclear HER4 seem to be co-located with ER, however, we did not find support for overall HER4 expression in independently predicting response of tamoxifen treatment. The possible influence of separate isoforms was not tested and future studies may further evaluate HER4 significance.

Reduced or absent Wwox expression has recently been associated with tamoxifen resistance in breast cancer and has also been proposed as a candidate predictive marker for treatment. We aimed to investigate the correlation of Wwox expression with the outcome of tamoxifen treatment by examining tissues from 912 randomized breast cancer patients. Paraffin-embedded tissues from patient tumors were arranged on tissue microarray, and Wwox protein was stained using immunohistochemistry. After microscopic examination, the results were analyzed with Cox regression, Kaplan-Meier survival curves and the log-rank test. In the group of cases having a tumor absent for Wwox expression, there was no difference in recurrence-free survival between treated and untreated patients (P=0.81). For treated cases with a tumor expressing moderate or strong Wwox protein, recurrence-free survival was improved (P=0.001 and P=0.003, respectively). The test for interaction between Wwox and treatment response demonstrated a decreased risk of recurrence for treated patients with a moderate or strong Wwox expression (HR=0.31, 95% CI 0.10-0.98 and HR=0.28, 95% CI 0.08-0.97, respectively). Our results indicate that patients with high expression of Wwox may gain more benefit from treatment with tamoxifen.

Aromatase is an important enzyme in the local synthesis of oestrogens and its expression has been shown to be increased in breast cancer through the activation of multiple promoters. However, the mechanisms behind this are not yet fully understood. A novel candidate in this context is the transcription factor forkhead box L2 (FOXL2), which has been recognised to be co-expressed with aromatase and transcriptionally active promoter II in developing goat and chicken ovaries. We propose that FOXL2 could be involved in the increased expression of aromatase in breast cancer. We examined FOXL2 and its relation to aromatase in 132 post-menopausal breast cancer patients by immunohistochemistry. Using in silico analysis, we further searched for FOXL2 binding-elements in the aromatase gene promoters. The results demonstrate that FOXL2 is expressed in breast cancer and influences clinical outcome with improved recurrence-free survival in cases with nuclear expression. In a multivariate Cox model, nuclear FOXL2 was a significant prognostic factor in ER-positive patients treated with tamoxifen (HR=0.18, 95% confidence interval (CI)=0.04-0.81, P=0.03). Tumours expressing nuclear FOXL2 were also more likely positive for stromal and/or cytoplasmic aromatase (P=0.03 and P=0.008, respectively). In silico analyses revealed binding elements of FOXL2 in promoters I.3, II and I.7 of the aromatase gene of which promoter I.7 was most significant. In conclusion, this is the first study to report that FOXL2 is expressed in breast cancer and correlates with aromatase as well as with clinical outcome. The results further strengthen a possible binding of FOXL2 to aromatase promoter I.7. Nevertheless, whether FOXL2 is a direct activator of aromatase requires further investigation.