2 REVIEW OF THE LITERATURE
2.7 Hyaluronan in Mesothelioma
2.7.2 Hyaluronan in lung mesothelioma and adenocarcinoma
2.7.1 Pathogenesis of pleural mesothelioma and lung adenocarcinoma
Malignant mesothelioma is one of the most highly malignant tumor types. It originates from serous mesothelial cells like those of pleura and peritoneum. Exposure to asbestos is the main risk factor for mesotheliomas. During the malignant transformation process the normal, flat epithelial cells of mesothelium are transformed into aggressive epithelioid or sarcomatoid cancer cells (Hjerpe and Dobra 2008). Experiments in vitro have shown that EGFR signaling has an important role in mesothelioma cell proliferation and migration (Jänne et al. 2002).
Lung cancer is one of the most common cancers. Lung cancers are divated into small cell carcinomas and non small cell carcinomas. Non small cell carcinomas are subdivated into adenocarcinomas, squamous cell carcinomas, large cell carcinomas, and sarcomatoid carcinomas (Petersen and Warth 2016). About one third of all lung cancers are adenocarcinomas, with several subtypes. Contrary to other lung cancers, patients with adenocarcinomas are typically non-‐‑smokers (Sun et al. 2007). The new classification of lung adenocarcinomas include preinvasive adenocarcinoma, minimally invasive adenocarcinomas and, invasive adenocarcinomas (Travis et al. 2011). Most of the lung adenocarcinomas arise from the epithelium of the terminal bronchiole or alveolar duct.
Normal bronchiole and the most adenocarcinomas express the thyroid transcription factor-‐‑
1 (TTF-‐‑1), while adenocarcinomas originating from the mucus-‐‑secreting epithelium are negative for TTF-‐‑1. Epidermal growth factor receptor mutation represents one of the molecular mechanisms and markers of lung adenocarcinomas (Pao et al. 2004).
2.7.2 Hyaluronan in lung mesothelioma and adenocarcinoma
Thylen and co-‐‑workers showed positive correlation between a short survival and hyaluronan content in mesothelioma patients (Thylen et al. 2001). All three HASs are expressed in mesotheliomas (Kanomata et al. 2005) and plasma membrane expression of CD44 has been reported in mesothelioma cells (Ordonez 2000). Affify et al showed intracellular staining of hyaluronan in mesothelioma cells and used it for the differentiation between lung adenocarcinoma and mesothelioma (Afify et al. 2005). Pleural hyaluronan level is a promising diagnostic marker for mesothelioma (Creaney et al. 2013).
The percentage of hyaluronan-‐‑positive cancer cells and their staining intensity is typically low in lung adenocarcinomas while tumor stroma is strongly positive (Pirinen et al. 2001).
A strong tumor cell-‐‑associated hyaluronan signal correlates with poor tumor differentiation, and a strong stromal signal is linked to the recurrence of the cancer and shortened survival (Pirinen et al. 2001). Hyaluronan binding protein 2 is overexpressed in lung adenocarcinomas (Wang et al. 2002). Functional studies suggest that lung adenocarcinoma progression is promoted by low molecular weight hyaluronan and hyaluronan binding protein 2 via the urokinase plasminogen activator pathway (Mirzapoiazova et al. 2015). Expression of CD44 and its variant forms in lung adenocarcinoma have been shown (Okudela et al. 2012, Pirinen et al. 2000) and high CD44 expression was shown to correlate with poor patient prognosis (Okueda et al. 2012).
Comparison of CD44 expression in mesotheliomas and different lung cancers indicated higher CD44 expression in mesotheliomas (Afify et al. 2005). The levels of standard CD44 and CD44v6 in the pleural fluid of mesothelioma patients are lower than in other lung malignancies (Porcel et al. 2011).
14 2.8 HYALURONAN IN DEVELOPMENT 2.8.1 Mouse development
Early development of all mammals is almost identical. After fertilization there are severals cleavage divisions. The 16-‐‑cell embryo is called morula, and in the morula stage all cells are still identical but then start to differentiate. One part of the cells form the embryo proper, while others take part in the implantation and later form the placenta. The cells in the embryo form two layers, called the epiblast and hypoblast. The epiblast layer is the origin of the cells of the developing animal and hypoblast layer forms extraembryonic mesoderm, and placenta with trophoblast cells. The epiblast cells form three different cell layers:
ectoderm, mesoderm and endoderm. Nervous and epidermal tissues originate from the ectoderm while the gut tube and its derivatives form the endoderm. All other tissues develop from the mesoderm. The disk-‐‑like embryo starts to elongate and polarize. The neural plate develops in the cranial end, and starts folding on day 7. The first somites appear in day 8, followed by the development of different organs in a specific sequence.
The heart tube starts to develop during day 8 and its endocardial cushions appear on day 10, and are finished on day 12. The first parts of the developing kidney appear on day 9.
The outmost layer of the embryonic ectoderm is the origin of the epidermis. Stratification of the epidermis starts in day 9, and hair follicle development on day 14. The stratum corneum is established during day 16 (Hardman et al. 1998). The whole development of mouse embryo takes 21 days (Kaufman 1992).
2.8.2 Hyaluronan in development
Hyaluronan is an important regulator throughout the development. It enables oocyte maturation and release during ovulation and regulates the penetration of spermatozoa to oocyte during fertilization (Richards 2005, Salustri et al. 1999). During the gastrulation hyaluronan has been suggested to facilitate the formation of the three-‐‑layered embryo (Müllegger and Lepperdinger 2002).
Hyaluronan accumulates in the heart tube of the developing embryo and is crucial for the heart valve development (Camenisch et al. 2000). It has been suggested, that hyaluronan is required also for epithelial tube formation in the developing prostate (Gakunga et al. 1997), and kidney (Pohl et al. 2000). During kidney development, hyaluronan promotes the branching of the ureteric bud (Pohl et al. 2000). Camenish et al demonstrated abnormalities in the development of the heart and vessels in HAS2 knockout mice. In particular, endocardial cushions did not develop correctly, probably accounting for the death of the embryo at E9.5 (Camenisch et al. 2000). This is consistent with the fact that during the formation of the heart valves HAS2 is upregulated, and is the main HAS isoform expressed in the heart tube (Tien and Spicer 2005). Hyaluronan content is also changed during the development of the cornea (Toole and Trelstad 1971), cartilage (Toole et al.
1972), joint cavity (Pitsillides et al. 1995) and palate (Pratt et al. 1973), and HAS2 expression is high in all of these locations (Tien and Spicer 2005). HAS2 overexpression showed shortened limbs in chick embryos. The morphology of the skeleton of the limbs was disordered, and some of its components had an abnormal location (Li et al. 2007a).
Additionally, HAS2 has a crucial role in chondrogenesis and chondrocyte differentiation (Li et al. 2007b, Matsumoto et al. 2009). HAS2 is probably the most important isoform during the development, responsible for hyaluronan production in the differentiation and growth of most organs. This is consistent with the findings that knockout of HAS1 or HAS3 or HAS1/HAS3 double knockouts do not show any developmental malformations.
During mouse skin development HAS2 mRNA is localized in the dermal area, and HAS3 mRNA in the epidermal compartment (Tien and Spicer 2005). However, HAS3 knockouts, or HAS3/HAS1 double knockouts have no defects in the skin during development,
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suggesting that HAS2 can replace HAS3 also in the developing epidermis (Tien and Spicer 2005). During epidermal stratification hyaluronan content is high in all layers of the epidermis, while during the formation of the stratum corneum, hyaluronan decreases, and eventually disappears from the terminally differentiated cells in both human and mouse skin (Tammi et al. 2005, Ågren et al. 1997a). The level of hyaluronan dramatically decreases after birth in the mouse epidermis. Correspondingly, HAS2 and HAS3 mRNA levels are lower in the newborn and adult mice as compared to the embryonic epidermal tissue
(Tammi et al. 2005).
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3 Aims of the study
Hyaluronan, a ubiquitous glycosaminoglycan of the extracellular matrix, accumulates particularly in situations with active cell renewal, like development, tissue healing and cancer. Despite recent progress on understanding its biology and functions, unsolved questions about its role in health and disease are still waiting for answers. For example, little is known about cellular and tissue localization of the HAS enzymes that are responsible for the synthesis of hyaluronan. The aim of this thesis work was to study the content and localization of hyaluronan, and proteins closely related to its production and functions, the hyaluronan synthases and the hyaluronan receptor CD44, by utilizing histochemical and immunohistochemical stainings. Developing tissues and cell cultures, UV induced cutaneous tumors, and the lung malignancies mesothelioma and adenocarcinoma, were used as examples.
The specific aims of this thesis were:
1. To investigate the distribution of HAS1-‐‑3 enzymes in developing mouse tissues, as well as their subcellular localization in cultured cells using immunohistochemistry and GFP-‐‑
tagged fusion proteins.
2. To study the content and changes of epidermal hyaluronan, CD44 and HAS1-‐‑3 in a mouse model of tumors induced by long-‐‑term exposure to UV radiation.
3. To compare and analyze the histochemical staining patterns of HAS1-‐‑3, CD44 and hyaluronan in pulmonary malignant mesotheliomas and adenocarcinomas.
collected and fixed, and 5 µμm thick sections were prepared for histological analysis. Human breast adenocarcinoma cells (MCF-‐‑7), human epidermal keratinocytes (HaCat) and human dermal fibroblasts were cultured as explained in publication I. UV-‐‑exposed skin samples for study II were gathered during the study of Kumlin et al. (Kumlin et al. 1998). The protocol for the preparation of the histological samples from skin is presented in study II.
The tumor samples for study III were collected in Oulu University hospital.
4.2 METHODS
4.2.1 EGFP-‐‑human Has1, 2, and 3 plasmid construction and transfection
The preparation of the human HAS1, 2, and 3 constructs is detailed in study I. Shortly, the open reading frame of each HAS was taken from human cDNA and amplified. The amplified HAS open reading frames were cloned to pEGFP-‐‑C1 vector. These plasmids were used for the transfections. The transfections were performed on cells cultured in 8-‐‑chamber slides (Ibidi GmbH, Martinsried, Germany) for microscopy and in 24-‐‑well plates (CELL STAR®, Greiner Bio-‐‑One, Kremsmunster, Austria) for the measurements of the hyaluronan concentrations in the growth medium.
4.2.2 Immunostaining
Live cells with the GFP-‐‑tagged HAS constructs were observed with a confocal microscope.
Cultured cells were fixed for the immunostainigs with 4% paraformaldehyde for 1 h in room temperature and permeabilized for 20 min with 1% BSA containing 0.1% Triton X100.
The cells were incubated overnight at 4°C with the HAS antibodies (Table 2), then washed and incubated with a fluorescein-‐‑labeled anti-‐‑goat secondary antibody. To perform double stainings for endoplamic reticulum, Golgi or hyaluronan, the HAS antibody was mixed with anti-‐‑Calnexin or anti-‐‑Golgi antibody, or bHABC (Table 2). The anti-‐‑ER and Golgi antibodies were visualized with a Texas Red anti-‐‑mouse secondary antibody, and hyaluronan with fluorescently labelled streptavidin. Biotinylated anti-‐‑goat secondary antibody (1:1000) with the avidin-‐‑biotin-‐‑peroxidase method was used for the wide-‐‑field microscopy.
HAS1, 2, and 3 immunostaining were used in the studies I, II and III. The tissue sections were incubated in 10 mM citrate buffer for 15 min at 120 °C in a pressure cooker. Non-‐‑
specific binding was blocked by incubation with 1% BSA and 0.1% gelatin in a phosphate buffer for 30 min. The sections were incubated overnight at 4°C with the hyaluronan synthase antibodies and biotinylated secondary antibodies and the avidin-‐‑biotin-‐‑
peroxidase method were used for their microscopic detection. Hyaluronan staining was used in studies I, II, and III. A complex containing the hyaluronan binding region of bovine articular cartilage aggrecan G1 domain and link protein (HABC) was biotinylated (bHABC)
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and used as a probe to detect hyaluronan (Tammi et al. 1994b). The immunostaining for CD44 was done in studies II and III. The tissue sections were first incubated in an antigen retrieval solution (Dako, Carpentia, CA, USA) for 30 min at 95°C (II) and then with an anti-‐‑
CD44 antibody (IM7) overnight at 4°C. There was no retrieval treatment in study III for the detection of CD44 with the Hermes 3 antibody. The blocking solution for CD44 stainings was 1% BSA in a phosphate buffer. The bound primary antibody was detected using a biotin-‐‑labeled secondary antibody and the avidin-‐‑biotin-‐‑peroxidase method. The antibodies and probes used are listed in Table 2.
The specificity of the HABC-‐‑probe was controlled by pretreating the samples with hyaluronidase or by blocking the probe with hyaluronan oligosaccharides. The antibodies for the HASs were controlled with several methods. The HAS-‐‑antibodies were omitted and replaced with non-‐‑immune goat IgG from incubation, and the antibodies were also treated with the peptides used in the immunization. Possible cross-‐‑reactivities of the HAS-‐‑
antibodies against other HAS family members were tested in transfected cells overexpressing different HASs (I, Figure 6). In controls for the CD44 stainings the primary antibodies IM7 (study II) and Hermes 3 (III) were omitted
Table 2 The antibodies and the probe used in the thesis
Calnexin 1:100 Cell Signaling Technology,
Inc., Boston, MA, USA
Golgin-‐‑97 1:100 Molecular Probes, Eugene,
OR, USA
Biotinylated anti-‐‑goat 1:1000 Vector Laboratories,
Burlingame, CA, USA
Biotinylated anti-‐‑mouse 1:100 Vector Laboratories,
Burlingame, CA, USA
Biotinylated anti-‐‑rat 1:100 Vector Laboratories,
Burlingame, CA, USA
Fluorescein anti-‐‑mouse 1:1000 Vector Laboratories,
Burlingame, CA, USA
Texas Red anti-‐‑mouse 1:1000 Vector Laboratories,
Burlingame, CA, USA
19 4.2.3 Microscopy
In study I the fluorescent images were obtained by a Zeiss Axio Observer inverted microscope equipped with a Zeiss LSM 700 confocal module (Carl Zeiss Microimaging GmbH, Jena, Germany). A conventional light microscope (Zeiss Axio Imager.M2 light microscope, Carl Zeiss) was used in studies I, II and III.
4.2.4 Analysis of hyaluronan concentration
Hyaluronan secretion into the culture medium (I) was measured with an ELSA assay as previously described (Hiltunen et al. 2002). Shortly, 96-‐‑well plates were precoated with HABC. The diluted samples and the hyaluronan standards were incubated in the wells for 1h. The hyaluronan attached was quantified with the sequential incubations of biotinylated HABC, horse radish streptavidin and the substrate-‐‑chromogen solutions containing 0.01 % of 3,3′,5,5′-‐‑tetramethybenzidine and 0.005 % H2O2 in a 0.1 M sodium acetate-‐‑1.5 mM citric acid buffer.
4.2.5 Evaluation and statistical analysis
The intensity of the HAS1-‐‑3, hyaluronan and CD44 stainings were analyzed by two independent evaluators in studies II and III. The statistical analysis was performed using the SPSS program (IBM Corporation, Armonk, New York, USA). The comparison of the extent and intensity of the stainings between treatments was done with the Kruskal-‐‑Wallis and Mann-‐‑Whitney U-‐‑tests (II). The correlations between the amount of epidermal hyperplasia and the hyaluronan and CD44 parameters were determined with Kendall’s test (II). In study III the differences between mesotheliomas and adenocarcinomas were calculated with the Pearson chi-‐‑square tests.
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5 Results
5.1 SUBCELLULAR LOCALIZATION OF HYALURONAN SYNTHASES (I)
5.1.1 Immunostainings of tissues
Because hyaluronan content of the dermis is high, human skin sections were used as an example to test the localization and amount of HAS isoenzymes in human tissues (I, Figure 5). Dermal fibroblasts were intensely stained with all HAS antibodies. Most of the staining detected was cytoplasmic, but also plasma membrane signal could be detected, for example for HAS1 (I, Figure 5b).
5.1.2 Immunostainings of cultured cells
We used three different cell lines, human skin dermal fibroblasts, human keratinocytes (HaCat) and transformed fibroblastic like COS-‐‑1 cells derived from monkey kidney, to study subcellular localization of endogenous HASs. These cell lines produce different amounts of hyaluronan, the fibroblasts producing the highest and COS-‐‑1 cells the lowest amount (I, table 1). In line with their high hyaluronan production (Jokela et al. 2013) fibroblasts were intensely stained for all HAS isoenzymes with the antibodies used (I, Figure 7a-‐‑c). The signal for HAS1 was low in HaCat keratinocytes, while HAS2 and HAS3 immunostainings were clearly positive, in accordance with the substantial levels of HAS2 and HAS3, and low HAS1 mRNA in these cells (Saavalainen et al. 2007) (I, Figure 7d-‐‑f). The immunostainings for all HASs were almost negative in COS-‐‑1 cells (I, Figure 7g-‐‑i).
HAS3 was abundant in plasma membrane and the protrusions of the cells. These areas were also rich in hyaluronan (I, Figure 8j-‐‑l). The majority of HAS1 was found intracellularly, mostly in the Golgi area. A weak signal was also seen in the plasma membrane and its protrusions (I, Figure 8a-‐‑c). Cytoplasmic vesicles contained most of the HAS2 staining (I, Figure 8d-‐‑f). Interestingly, a part of the HAS2 signal was also localized in the ER and nuclear membrane, especially in the fibroblasts (I, Figure 7b).
Native MCF-‐‑7 cells, expressing a low level of HAS3 mRNA, a modest level of HAS2, and almost no HAS1, produce about 2.6 ng hyaluronan/10,000cells/24h (Kultti et al. 2009), while the MCF-‐‑7 cells transfected with HAS1-‐‑3 constructs synthesize large quantities of hyaluronan (Kultti et al. 2009). The antibody for HAS1 stained the HAS1 overexpressing cells nicely, but produced no signal in cells overexpressing HAS2 and HAS3 (I, Figure 6).
Likewise, the antibody for HAS2 showed no cross-‐‑reaction in cells overexpressing HAS1 and HAS3. No cross-‐‑reactivity was seen with HAS3 antibody either.
5.1.3 GFP-‐‑tagged HAS proteins
To confirm the different subcellular distributions of the HAS isoforms, as suggested by the immunostainings, MCF-‐‑7 cells were transiently tranfected with the human HAS-‐‑GFP-‐‑
constructs. Each of the GFP-‐‑tagged isoenzymes had a typical subcellular distribution (I, Figure 9). GFP-‐‑HAS1 showed the lowest signal in the plasma membrane. It was especially abundant in the Golgi area and in the ER. However, some GFP-‐‑HAS1 positive intracellular vesicles were seen near the plasma membrane and in the thin protrusions of the plasma membrane (I, Figure 9a,b). The GFP-‐‑HAS2 signal was primarily found in the ER, Golgi and
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cytoplasmic vesicles. Some of the protrusions of the plasma membrane, especially those standing on the edges of lamellipodia, contained high levels of GFP-‐‑HAS2 (I, Figure 9c,d).
GFP-‐‑HAS3 had the highest plasma membrane signal among the HASs, in addition to the intracellular locations similar to the other HAS isoenzymes. The apical surface of the GFP-‐‑
HAS3 expressing cells was covered by very long plasma membrane protrusions positive for GFP-‐‑HAS3 (I, figure 9e,f).
5.2 SPATIAL AND TEMPORAL DISTRIBUTION OF HYALURONAN AND HAS1-3 DURING MOUSE EMBRYONIC DEVELOPMENT (I)
Paraffin sections from mouse embryos at different ages were stained with bHABC and HAS antibodies to detect hyaluronan and HAS1-‐‑3. The specificity of the hyaluronan staining was controlled by treatment of the sections with hyaluronidase, and blocking the probe with hyaluronan oligosaccharides. The spesificities of the HAS1-‐‑3 stainings were determined by blocking with peptides corresponding to those used for immunization (I, Figure2, i-‐‑l) or replacing the primary antibody with non-‐‑immune IgG.
The stainings for hyaluronan were typically intense in all stages of the development (I, Figures 1-‐‑4). Hyaluronan was abundant especially in the tissues of mesodermal origin, like the stuctures surrounding the neural tube, branchial arch, cardic tube and its cushions in samples from the E9 embryos (I, Figure 1a-‐‑d). During the embryonic day11 (E11)-‐‑E15 stage HA accumulated in the mesenchymal tissues all over the body (I, Figures 2m, 3a), in the skin and its underlying connective tissues (I, Figure 2e, Figure 3a,c), cartilage (I, Figure 3m) and certain brain areas (I, Figure 3a,q). In the E17 stage hyaluronan stainings were still intense in the connective tissues, part of the brain, kidney and developing eye (I, Figure 4a,e,I,m). Interestingly, at this stage parts of the brain, liver and calcified bones were almost negative for hyaluronan (Figure 4).
As expected, the HAS stainings in the embryos were localized in the cells (as opposed to the extracellular matrix). The main location was cytoplasm for all HASs (I, Figures 1-‐‑4).
Nevertheless, HAS2 signal was seen in positions consistent with plasma membrane in the tubular epithelium of the developing kidneys (I, Figure 4o). In addition, some plasma membrane protrusions on the mesenchymal cells of the intramembraneous bones were stained with the HAS1 and HAS3 antibodies (I, Figures 2n,p, arrows). In general, the mesenchymal cells in particular were highly positive for all HASs (I, Figures 2n-‐‑p), but the staining was also high in the developing epidermal keratinocytes. In the kidney, the stainings of the HASs were most intense in the tubular cells (I, Figures 4n-‐‑p). The sections through a whole E17 mouse embryo stained for HAS1, 2 and 3 and hyaluronan, show a relatively high signal intensity of all HASs e.g. in the developing skin and cartilage, as well as the overall distribution of each isoenzyme (Figure 4). Large amounts of hyaluronan are found especially in the connective tissues, brain and kidney (I: Figure 4).
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Figure 4. Distribution of hyaluronan and hyaluronan synthases in sections of a whole mouse E17 embryo. The sections were stained with bHABC to detect hyaluronan and with affinity purified polyclonal antibodies for HAS1, HAS2 and HAS3. The brown color indicates the signal for hyaluronan and the antibodies, and hematoxylin was utilized to stain the nuclei (blue). Magnification bar 1 mm.
The staining intensity of HAS2 was generally highest in the E9 embryos, but signals were present also for HAS1 and HAS3 (I, Figure 1). On the embryonic day 11 all HASs stained with high intensity in the heart, and mesenchymal tissues such as dermis. At this stage, a
The staining intensity of HAS2 was generally highest in the E9 embryos, but signals were present also for HAS1 and HAS3 (I, Figure 1). On the embryonic day 11 all HASs stained with high intensity in the heart, and mesenchymal tissues such as dermis. At this stage, a