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Ectopic Expression of Sortilin 1 (NTR-3) in Patients with Ovarian Carcinoma.

Gene expression profiling of ovarian carcinoma tissues has shown an increase of four-fold expression of SORT1 gene. Sortilin 1 (NTR-3) is a 95–100 kDa protein normally expressed in heart, brain, placenta, skeletal muscle, spinal cord, thyroid, and testis. However, its expression has never been reported in normal ovary. Here, we report expression of sortilin 1 in ovarian carcinoma tissues both at gene and protein levels. Sortilin 1 was expressed in all ovarian carcinoma patients (n=15) as well as ovarian carcinoma cell lines (n=5) regardless of their phenotypic characteristics. Non-malignant ovaries (n=6) did not express sortilin 1. The molecular basis for this ectopic expression is not yet clear. Our results showed a major cell surface expression of sortilin 1 rather than ER-Golgi compartment where it is mainly expressed. This finding may introduce sortilin 1 as a novel tumor marker for diagnosis of ovarian carcinoma and may signify its therapeutic value in targeted therapy.


Introduction
Ovarian cancer is one of the leading causes of cancer deaths in women. Due to the lack of any specific tumor marker, the diagnosis of this disease usually happens in advanced stages leading to high mortality rate. Although, the increased level of CA125 is considered to be a diagnostic marker (in about 80% of cases), but it is still not reliable and usually increases at very late stages. Since no suitable diagnostic and curative therapy is available, there is a high demand for finding novel tumor markers in this malignancy both for diagnostic and therapeutic purposes.
Gene expression profiling of ovarian carcinoma patients in early and late stages or with low malignant potential has shown a nearly four-fold increase of SORT1 gene expression (1) .
The human SORT1 gene consists of 22 exons located on the short arm of chromosome 1 (1p21.3-p13.1) encoding a type I receptor of 831 amino acids with a molecular weight of 95-100 kDa. Seven non-coding transcript variants for this gene have also been reported. SORT1 is expressed at gene level in heart, brain, placenta, skeletal muscle, spinal cord, thyroid, and testis (2) . Its expression, however, has never been reported in normal ovary. Sortilin 1 is a member of family of Vps10pdomain receptors. It is known as a multiligand molecule and such proteins as lipoprotein lipase (LPL), Neurotensin 3 (NT3), Receptor associated protein (RAP), proNGF, and thyroglobulin are among its potential ligands (2,(3)(4)(5) .
The function of sortilin 1 varies depending on its location and the presence of its ligands. Sortilin 1 acts as a sorting receptor when it is expressed in trans-golgi network and also as a clearance receptor when it is expressed on cell surface (2,(3)(4)(5) .
The role of sortilin 1 and NT3 as its ligand in cancer cell growth has previously been reported (6) . Although, sortilin 1 as a type I receptor lacks the necessary domains involved in signaling, but is an essential part of a receptor complex for pro-NGF induced neural cell death (5,7) .
The four-fold overexpression of SORT1 in ovarian carcinoma together with its driving role in cancer cell growth brings forward the potential involvement of this protein in the pathobiology of this disorder. To test this hypothesis, we studied the expression of sortilin 1 in 15 ovarian carcinoma patients both at gene and protein levels. Sortilin 1 was expressed in all 15 ovarian carcinoma tumor tissues as well as related cell lines, while no expression of this molecule in non-malignant ovarian tissue was found. The expression of sortilin 1 in ovarian carcinoma patients may represent a crucial role of this protein in tumorigenesis driving machinery.

Patients and tissue samples
Ovarian tissue samples were obtained from patients at Imam Khomeini Hospital. After a consent letter was signed by each patient, all the aspects of this study were approved by the Avicenna Local Ethics Committee. After surgical resection, each fresh tumor specimen was immediately divided into two portions: one portion was instantly frozen in liquid nitrogen and the other was OCT (Optimal Cutting Compound) embedded using Jung freezing medium (Leica Instruments, Nussloch, Germany) for immunohistochemical investigations.

RNA extraction and cDNA synthesis
Total RNA was extracted from cell lines and tissue samples using RNA-Bee reagent (BioSite, Täby, Sweden) according to the manufacturer's instruction. The quality of the RNA samples was determined by agarose gel electrophoresis after staining with ethidium bromide, and visualization under UV light. Total RNA was unfolded at 65 °C for 10 min. cDNA was then synthesized using 5 µg of total RNA in 20 µL reaction mixture consisting of 4 µL of 5x reaction buffer, 1 µL of 10 mM dNTPs, 1 µL of 10 pmol/mL random hexamers (N6), and 200 U M-MuLV reverse transcriptase (Fermentas GmbH, St. Leon-Rot, Germany). The reaction mixture was incubated at 42 °C for 45 min.
Due to the poor quality of RNA prepared from some tissue samples, nested-PCR was carried out for detection of SORT1 gene in ovary tissues using "GTTAACAGCAGAG GTGTCTGGAG" as sense primer and "AA ACATACTGCTTTGTGGATTTC" as antisense primer. Briefly, 1 μl of RT-PCR product was added to a new 24 μl reaction mixture containing 2.5 μl 10x PCR buffer, 3 μl 25 mM MgCl2, 1 μl 10 mM dNTPs, 5 pmol of each primer, and 1 Unit Taq DNA polymerase. Nested PCR was performed for 20 cycles generating an amplicon of 225 bp. PCR products were finally visualized by running agarose gel electrophoresis containing ethidium bromide.
To verify the relative amounts of protein in each lane, the level of β-actin as an internal control was measured with anti-β-actin monoclonal antibody (Sigma). Membranes were developed using the ECL detection system (GE Healthcare, Uppsala, Sweden) according to the manufacturer's instruction.

Immunohistochemistry (IHC)
Five μm sections of OCT-embedded ovary tissues were cut on a cryostat (Thermo-Shandon, UK), dried overnight, fixed in acetone for 2 min at -20 °C and kept at -70 °C until use. Slides were then thawed and washed three times (3×3 min) with Tris-Buffered Saline, pH 7.4 containing 5% bovine serum albumin (TBS-BSA). At the next step, the slides were blocked with 5% goat serum diluted in TBS-BSA for 30 min and incubated with 1:500 diluted primary anti-sortilin 1 polyclonal antibody (Abcam, Cambridge, UK) overnight at 4 °C. To confirm the specificity of the IHC staining, negative reagent control slides were incubated with TBS-BSA without any primary antibody (no-antibody control) or with pre-immune rabbit serum with the same IgG concentration. Following three washes, the slides were then incubated with 1:75 dilution of fluorescin isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Ray Biotech, Inc. GA, USA) for 45 min.

SORT1 gene expression
Representative RT-PCR gel of ovarian carcinoma patients is shown in Figure 1. All ovarian carcinoma patients (n=15) expressed SORT1 at the mRNA level (Table 1). SORT1 was also expressed in all six ovarian carcinoma cell lines (Table 2). Non-malignant ovary, however, failed to express this gene (data not shown).

Sortilin 1 protein expression
Western blot analyses of six ovarian carcinoma cell lines showed a band of 95-100 kDa as well as an extra band of 50 kDa (Figure 2A). Cell lysates from primary ovarian carcinoma tissues of different subtypes showed only a band of 95-100 kDa  Figure 2B). None of the nonmalignant ovary samples expressed sortilin 1 at protein level ( Figure 2C).
To confirm the expression and localization of sortilin 1 in tumor samples, we performed indirect immunofluorescence staining on 7 malignant ovarian tissue samples and 5 normal ovaries. The positive staining was only observed in the cell membrane of malignant epithelial cells. Consistent with the results obtained by Western blotting, the ovarian carcinomas (four of seven specimens) expressed sortilin 1 mainly localized to cell surface rather than ER-Golgi compartment in immunohistochemistry & tainins. Three out of seven ovarian carcinoma samples showed lower levels of sortilin 1 in immunofluorescence experiments. Similar to Western blot results, no sortilin 1 expression was observed in normal ovary tissue sections. One representative out of seven immunofluorescence experiments is shown in Figure 3.

Discussion
The functional roles of neuropeptides (NT) and their receptors in cancer cell growth are now well established. The growth effect of sortilin 1 as neurotensin-3 receptor (NTR-3) in prostate, colon, and pancreatic cancer cell lines strongly suggests the involvement of this protein in cell growth (8) . Sortilin 1 was also expressed in a panel of breast cancer cell lines (our unpublished data). It seems that the expression of sortilin 1 is not restricted to ovarian carcinoma and it might be also expressed in other solid tumors. This may signify more important function of this receptor in cancer development.
In our study, the cell lysates from all ovarian cancer tissues and cell lines but not from non-malignant ovary tissues, showed expression of 95-100 kDa sortilin 1 protein indicating an ectopic expression. Interestingly, a 50 kDa band was only observed in ovarian carcinoma cell lines whereas it was absent in breast cancer cell lines (data not shown). This 50 kDa band may represent an unidentified isoform of sortilin 1 which requires further characterization.
Although, sortilin 1 is mainly localized to ER-Golgi apparatus, it is also expressed as a mature cell surface receptor (9) . Our immunofluorescence experiments showed that sortilin 1 is mainly expressed on tumor cell surface rather than ER-Golgi compartment. The surface expression of this protein may provide a more significant role for this protein in ERK signaling pathway (10) leading to proliferation   T7, T10, T14, T18, T19, T25, and T32 corresponding to patients in Table I selected based on their differentiation. Human brain (HB) cell lysate served as positive control. C) Lanes 1-6 representing non-malignant ovarian tissue samples. Lane 7 represents human brain cell lysate. β-actin expression was used as an internal loading control of cancer cells. Apparently, expression of sortilin 1 in ovarian cancer cells as a cell surface receptor does not induce apoptosis excluding its proapoptotic activity. This might be due to the lack of other co-receptors required for triggering apoptosis in ovary cancer cells and the absence of their signaling mediators (proNGF, proBDNF). It could be also due to structural differences in sortilin 1 expressed by cancer cells leading to impaired heterodimerization with other co-receptors (p75NTR) which is necessary for signal transduction. In general, the dual functional proapoptotic and anti-apoptotic roles of sortilin 1 must be considered in normal and pathogenic conditions.
Among patients enrolled in this study two patients had tumors with morphology of nonepithelial origin indicating that expression of sortilin 1 is not restricted to epithelial cells. Due to the lack of patient samples from early stages and also limited number of patients, it was not possible to correlate the expression level of sortilin 1 to the stage of the disease as well as to CA125 level or age of the patients. In this regard the expansion of the study in more patients is warranted.
Recent report shows that sortilin 1 is also expressed in thyroid epithelial cells capable of binding with high affinity to thyroglobulin, another evidence for sortilin 1 as a multiligand receptor (4) . This finding together with the expression of sortilin 1 in ovarian carcinoma may explain the 4% incidence of metastasis of ovarian carcinoma to thyroid glands, rich in thyroglobulin (11) as well as the same incidence in brain metastases (12) .
Altogether, ectopic expression of sortilin 1in ovarian carcinoma tissues, its absence in normal human ovary, its involvement in ERK signaling pathway (10) as well as its restricted tissue expression in women makes it a potential candidate for developing more selective targeted therapy approach in patients with ovarian carcinoma. In this regard, generation of monoclonal antibodies capable of inducing apoptosis and also competing with sortilin 1 ligand binding site (Vps10p domain) is recommended.
Women with cancer may suffer from infertility after medical treatment. The current approach to protect reproductive capability in women with cancer is ovarian cortex cryopreservation prior to any medical treatment and re-implanting the frozen ovary tissues after cancer therapy. Detection of sortilin 1, as a unique marker in the cryopreserved tissue for distinguishing malignant ovaries from non malignant ones before reimplantation may decrease the risk of relapse in these patients leading to regaining their reproductive capability.
To the best of our knowledge, this study is the first report describing ectopic expression of sortilin 1 in patients with ovarian carcinoma. Further functional impacts and structural characterization are needed to explore its potential role in the pathogenesis of ovarian carcinoma.