Kirsi Hämäläinen

High numbers of macrophages, especially M2-like (CD163- positive), correlate with hyaluronan accumulation and poor outcome in breast cancer

High amounts of tumour‐associated macrophages (TAMs) and hyaluronan (HA) correlate with tumour aggressiveness in breast cancer, but the relationship between these parameters is unclear. The aim of this study was to assay the numbers of TAMs in 278 human breast cancer cases, and their correlations with HA‐related factors, clinical variables, and outcome.

Expression of Hyaluronan Synthases (HAS1–3) and Hyaluronidases (HYAL1–2) in Serous Ovarian Carcinomas: Inverse Correlation between HYAL1 and Hyaluronan Content

BackgroundHyaluronan, a tumor promoting extracellular matrix polysaccharide, is elevated in malignant epithelial ovarian tumors, and associates with an unfavorable prognosis. To explore possible contributors to the accumulation of hyaluronan, we examined the expression of hyaluronan synthases (HAS1, HAS2 and HAS3) and hyaluronidases (HYAL1 and HYAL2), correlated with hyaluronidase enzyme activity hyaluronan content and HAS1–3 immunoreactivity.MethodsNormal ovaries (n = 5) and 34 serous epithelial ovarian tumors, divided into 4 groups: malignant grades 1+2 (n = 10); malignant grade 3 (n = 10); borderline (n = 4) and benign epithelial tumors (n = 10), were analyzed for mRNA by real-time RT-PCR and compared to hyaluronidase activity, hyaluronan staining, and HAS1–3 immunoreactivity in tissue sections of the same specimens.ResultsThe levels of HAS2 and HAS3 mRNA (HAS1 was low or absent), were not consistently increased in the carcinomas, and were not significantly correlated with HAS protein or hyaluronan accumulation in individual samples. Instead, the median of HYAL1 mRNA level was 69% lower in grade 3 serous ovarian cancers compared to normal ovaries (P = 0.01). The expression of HYAL1, but not HYAL2, significantly correlated with the enzymatic activity of tissue hyaluronidases (r = 0.5; P = 0.006). An inverse correlation was noted between HYAL1 mRNA and the intensity of hyaluronan staining of the corresponding tissue sections (r = -0.4; P = 0.025).ConclusionThe results indicate that in serous epithelial ovarian malignancies HAS expression is not consistently elevated but HYAL1 expression is significantly reduced and correlates with the accumulation of hyaluronan. (233 words)

Antiangiogenic Gene Therapy With Soluble VEGFR-1, -2, and -3 Reduces the Growth of Solid Human Ovarian Carcinoma in Mice

We studied antiangiogenic and antilymphangiogenic effects of sVEGFR-1 (sFlt-1), sVEGFR-2 (sFlk-1/KDR), and sVEGFR-3 (sFlt-4) gene transfers and their combinations in intraperitoneal ovarian cancer xenograft mice (Balb/c-Anu, n = 55). Gene therapy was initiated when the presence of sizable tumors was confirmed in magnetic resonance imaging (MRI). Adenovirus-mediated gene transfer was performed intravenously via tail vein as follows: AdLacZ as a control (group I), AdsFlt-1 (group II), AdsKDR (group III), AdsFlt-4 (group IV) and two combination groups of AdsFlt-1 and AdsFlt-4 (group V) and AdsFlt-1, AdsKDR, and AdsFlt-4 (group VI). Antitumor effectiveness was assessed by sequential MRI, immunohistochemistry, microvessel density, overall tumor growth, and survival time. In combination group VI, intraperitoneal tumors were significantly smaller than in the control group at the end of the follow-up (P < 0.001). Furthermore, in group VI the microvessel density (microvessels/mm(2)) in tumor tissue and the total area of tumors covered by microvessels were significantly smaller than in the controls. One mouse in group V was cured. The combined antiangiogenic gene therapy with soluble VEGFRs reduced tumor growth, tumor vascularity, and ascites formation in ovarian cancer xenografts. The results suggest that the combined antiangiogenic gene therapy is a potential approach for the treatment of ovarian cancer patients.

Hyaluronan synthases (HAS1-3) and hyaluronidases (HYAL1-2) in the accumulation of hyaluronan in endometrioid endometrial carcinoma

BackgroundHyaluronan accumulation correlates with the degree of malignancy in many solid tumor types, including malignant endometrial carcinomas. To elucidate the mechanism of hyaluronan accumulation, we examined the expression levels of the hyaluronan synthases (HAS1, HAS2 and HAS3) and hyaluronidases (HYAL1 and HYAL2), and correlated them with hyaluronan content and HAS1-3 immunoreactivity.MethodsA total of 35 endometrial tissue biopsies from 35 patients, including proliferative and secretory endometrium (n = 10), post-menopausal proliferative endometrium (n = 5), complex atypical hyperplasia (n = 4), grade 1 (n = 8) and grade 2 + 3 (n = 8) endometrioid adenocarcinomas were divided for gene expression by real-time RT-PCR, and paraffin embedded blocks for hyaluronan and HAS1-3 cytochemistry.ResultsThe mRNA levels of HAS1-3 were not consistently changed, while the immunoreactivity of all HAS proteins was increased in the cancer epithelium. Interestingly, HAS3 mRNA, but not HAS3 immunoreactivity, was increased in post-menopausal endometrium compared to normal endometrium (p = 0.003). The median of HYAL1 mRNA was 10-fold and 15-fold lower in both grade 1 and grade 2+3 endometrioid endometrial cancers, as compared to normal endometrium (p = 0.004-0.006), and post-menopausal endometrium (p = 0.002), respectively. HYAL2 mRNA was also reduced in cancer (p = 0.02) and correlated with HYAL1 (r = 0.8, p = 0.0001). There was an inverse correlation between HYAL1 mRNA and the epithelial hyaluronan staining intensity (r = -0.6; P = 0.001).ConclusionThe results indicated that HYAL1 and HYAL2 were coexpressed and significantly downregulated in endometrioid endometrial cancer and correlated with the accumulation of hyaluronan. While immunoreactivity for HASs increased in the cancer cells, tumor mRNA levels for HASs were not changed, suggesting that reduced turnover of HAS protein may also have contributed to the accumulation of hyaluronan.

Nuclear expression of Snail1 in borderline and malignant epithelial ovarian tumours is associated with tumour progression

BackgroundTranscription factor Snail1 has a central role in induction of epithelial-mesenchymal transition (EMT). The aim of the present study was to elucidate the expression of Snail1 protein during epithelial ovarian tumourigenesis and to study the association of Snail1 expression with clinicopathological factors and prognosis.MethodsEpithelial and stromal fibroblast-like fusiform cells of 14 normal ovarian samples, 21 benign, 24 borderline and 74 malignant epithelial ovarian tumours were studied for Snail1 protein using immunohistochemistry.ResultsNuclei of surface peritoneal cells of normal ovaries (n = 14) were regarded as negative for Snail1. Nuclear expression of Snail1 protein in epithelial ovarian tumours was increased during tumour progression from precursor lesions into carcinomas both in epithelial (p = 0.006) and stromal cells (p = 0.007). Nuclei of benign tumours (n = 21) were negative for Snail1. In borderline tumours (n = 24) occasional positive epithelial cells were found in 2 (8%) samples and in 3 (13%) samples stromal cells were focally positive for Snail1. In carcinomas (n = 74) focal Snail1 staining in epithelial cells was present in 17 (23%) tumours, and in stromal cells in 18 (24%) tumours. Nuclear expression of Snail1 in epithelial or stromal cells was not associated with clinicopathological factors or prognosis.ConclusionNuclear Snail1 expression seems to be related to tumour progression, and expression in borderline tumours indicates a role for Snail1 in early epithelial ovarian tumour development. Snail1 also appears to function more generally in tissue remodelling as positive staining was demonstrated in stromal cells.

Antiangiogenic gene therapy with soluble VEGF‐receptors ‐1, ‐2 and ‐3 together with paclitaxel prolongs survival of mice with human ovarian carcinoma

We compared effects of antiangiogenic gene therapy with a combination of soluble sVEGFR‐1, sVEGFR‐2 and sVEGFR‐3 to chemotherapy with carboplatin and paclitaxel and to antiangiogenic monoclonal anti‐VEGF‐antibody bevacizumab in an intraperitoneal ovarian cancer xenograft model in mice (n = 80). Gene therapy was also combined with chemotherapy. Therapy was initiated when sizable tumors were confirmed in magnetic resonance imaging (MRI). Adenovirus‐mediated gene transfer was performed intravenously (2 × 109 pfu), while chemotherapy and monoclonal anti‐VEGF‐antibody were dosed intraperitoneally. The study groups were as follows: AdLacZ control (n = 21); combination of AdsVEGFR‐1, ‐2 and ‐3 (n = 21); combination of AdsVEGFR‐1, ‐2, ‐3 and paclitaxel (n = 9); bevacizumab (n = 14); paclitaxel (n = 9) and carboplatin (n = 5). Effectiveness was assessed by survival time and surrogate measures such as sequential MRI, immunohistochemistry, microvessel density and tumor growth. Antiangiogenic gene therapy combined with paclitaxel significantly prolonged the mean survival of mice (25 days) compared to the controls (15 days) and all other treatment groups (p = 0.001). Bevacizumab treatment did not have any significant effect on the survival. Tumors of the mice treated by gene therapy were significantly smaller than in the control group (p = 0.021). The mean vascular density and total vascular area were also significantly smaller in the tumors of the gene therapy group (p = 0.01). These results show potential of the antiangiogenic gene therapy to improve efficacy of chemotherapy with paclitaxel and support testing of this approach in a phase I clinical trial for the treatment of ovarian cancer.

Reduced γ-Catenin Expression and Poor Survival in Oral Squamous Cell Carcinoma

OBJECTIVE To investigate whether reduced expression of alpha-, beta-, or gamma-catenin predicts poor survival in oral squamous cell carcinoma (OSCC). DESIGN Immunohistochemical analyses of a retrospective cohort. SETTING University-affiliated hospital. PATIENTS One hundred twenty-four patients with OSCC. MAIN OUTCOME MEASURE The prognostic value of gamma-catenin expression on disease-specific survival in different T and N category groups in patients with OSCC. RESULTS Reduced expression of gamma-catenin correlated with poor tumor differentiation of OSCC (P = .04). Patients with reduced gamma-catenin expression in the primary tumor had significantly more frequent lymph node metastasis than did patients with normal gamma-catenin expression (P = . 03). Reduced expression of gamma-catenin (004) but not of alpha-catenin (P = .25) or beta-catenin (P = .48) correlated with poor clinical outcome. Reduced gamma-catenin expression predicted poor disease-specific survival also in the 92 patients with T1 or T2 tumors (P = . 02). In multivariate analysis, advanced T category (P = . 04), neck lymph node metastases (P = . 01), and reduced gamma-catenin expression (P = . 05) were independently related to poor survival. CONCLUSIONS Reduced expression of gamma-catenin was associated with poor differentiation of OSCC, with neck lymph node metastases, and, more importantly, with poor disease-specific survival. Loss of gamma-catenin expression seems to contribute to metastatic properties of OSCC. Evaluation of the expression pattern of gamma-catenin may be useful for predicting outcome in patients with OSCC.

Cotargeting of VEGFR-1 and -3 and angiopoietin receptor Tie2 reduces the growth of solid human ovarian cancer in mice

Despite optimal surgery and chemotherapy, the prognosis of ovarian cancer patients remains poor and new treatments are urgently needed. Solid tumors require the formation of new vessels for growth and metastasis. In the present study, we have used soluble vascular endothelial growth factor (sVEGF) receptors sVEGFR-1 and -3, soluble receptors Tie1 and Tie2 and their combinations in an ovarian cancer xenograft model. Human ovarian cancer cells were injected intraperitoneally into nude mice (n=42) and magnetic resonance imaging (MRI) was used for confirming tumors before gene delivery. Treatment with combined AdsVEGFR-1, AdsVEGFR-3 and AdsTie2 significantly decreased the size of the intraperitoneal tumors compared with the controls (AdLacZ; P=0.038) with significantly less microvessels and vascular area. Unexpectedly, treatment with combined AdsTie1 and AdsTie2 led to a dramatic shortening of the survival which was not observed in the groups receiving either of the soluble receptors alone (P=0.031). The only difference to other treatments was liver toxicity observed after the combined Tie receptor treatment. In conclusion, combined inhibition of VEGFR-1, VEGFR-3 and Tie2 pathways was safe and provided efficient therapy for ovarian cancer in mice.

Preclinical Safety, Toxicology, and Biodistribution Study of Adenoviral Gene Therapy with sVEGFR-2 and sVEGFR-3 Combined with Chemotherapy for Ovarian Cancer

Abstract Antiangiogenic and antilymphangiogenic gene therapy with soluble vascular endothelial growth factor receptor-2 (VEGFR-2) and soluble VEGFR-3 in combination with chemotherapy is a potential new treatment for ovarian carcinoma. We evaluated the safety, toxicology, and biodistribution of intravenous AdsVEGFR-2 and AdsVEGFR-3 combined with chemotherapy in healthy rats (n=90) before entering a clinical setting. The study groups were: AdLacZ and AdLacZ with chemotherapy as control groups, low dose AdsVEGFR-2 and AdsVEGFR-3, high dose AdsVEGFR-2 and AdsVEGFR-3, combination of low dose AdsVEGFR-2 and AdsVEGFR-3 with chemotherapy, combination of high dose AdsVEGFR-2 and AdVEGFR-3 with chemotherapy, and chemotherapy only. The follow-up time was 4 weeks. Safety and toxicology were assessed by monitoring the clinical status of the animals and by histological, hematological, and clinical chemistry parameters. For the biodistribution studies, quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used. Low dose (2×10(10) vp) AdsVEGFR-2 and AdsVEGFR-3 gene therapy was well tolerated, even when gene therapy was combined with chemotherapy. Notably, only transient elevation of liver enzymes and mild regenerative changes were seen in liver after the gene transfer in the groups that received high doses (2×10(11) vp) of AdsVEGFR-2 and AdsVEGFR-3 with or without chemotherapy. No life-threatening adverse effects were noticed in any of the treatment groups. The highest protein concentration of soluble VEGFR-2 (sVEGFR-2) in circulation was seen 1 week after the gene transfer. The combination of chemotherapy to gene therapy seemed to prolong the time of detectable transgene protein at least 1 week in the circulation. The expression of AdsVEGFR-2 and AdsVEGFR-3 transgenes was mainly seen in the liver and spleen as detected by qRT-PCR. According to these results, AdsVEGFR-2 and AdsVEGFR-3 gene therapy combined with chemotherapy is safe and can be brought to clinical testing in ovarian cancer patients.

High extent of O-GlcNAcylation in breast cancer cells correlates with the levels of HAS enzymes, accumulation of hyaluronan, and poor outcome

PurposeObesity and oversupply of glucose, e.g., due to nutritional factors may shape the tumor microenvironment favorable for tumor progression. O-GlcNAcylation, a reversible modification of intracellular proteins, influences on several cellular functions and is connected to many diseases including cancer. Glycosaminoglycan hyaluronan (HA) enhances tumor progression and in breast cancer HA accumulation associates strongly with poor outcome. In vitro studies have suggested that O-GlcNAcylation may enhance HA synthesis. The aim of this study was to investigate the correlations between O-GlcNAcylation, HA-related parameters, and disease outcome in a clinical breast cancer material consisting of 278 breast cancer cases.MethodsIn microscopic analyses, O-GlcNAc staining of the breast carcinoma cells was evaluated in several randomly picked high-power fields of each section. The extent of cytoplasmic O-GlcNAc staining was graded as either low or high according to the intensity of the staining and the percentage of stained cells. The extent of nuclear O-GlcNAc staining was categorized as either low or high according to the percentage of stained nuclei.ResultsA high extent of both cytoplasmic and nuclear O-GlcNAcylation correlated with an increased relapse rate, development of distant metastases, and poor outcome. A high extent of cytoplasmic O-GlcNAcylation correlated also with the accumulation of all hyaluronan synthase (HAS1-3) proteins and with a large amount of HA in the tumor stroma. In addition, a high extent of nuclear O-GlcNAcylation associated with obesity.ConclusionsThe results suggest a mechanistic association between increased O-GlcNAcylation and HA synthesis, leading to a HA-rich microenvironment favorable for breast cancer progression.