Surgical Pathology and Cytopathology Unit, Department of Medicine, University of Padua, Padua, Italy
Corresponding author: Ambrogio Fassina, MD, Surgical Pathology and Cytopathology Unit, Department of Medicine, University of Padua, Via Aristide Gabelli, 61, 35121-Padua, Italy; Fax: (011) 39 049 827 3782; email@example.com
Ovarian serous carcinoma (OSC) is a fatal gynecologic malignancy usually presenting with bilateral localization and malignant peritoneal effusion. Programmed cell death 4 (PDCD4) is a tumor suppressor gene whose expression is directly controlled by microRNA-21 (miR-21). Exosomes are small cell-derived vesicles that participate in intercellular communication, delivering their cargo of molecules to specific cells. Exosomes are involved in several physiological and pathological processes including oncogenesis, immunomodulation, angiogenesis, and metastasis. The current study analyzed the expression of PDCD4 and miR-21 in resected OSC specimens and in cells and exosomes from OSC peritoneal effusions.
PDCD4 was immunohistochemically examined in 14 normal ovaries, 14 serous cystadenoma (CA), and 14 OSC cases. Quantitative reverse transcriptase-polymerase chain reaction analysis of PDCD4 and miR-21 expression was performed in CA and OSC cases and in cells and exosomes obtained from 10 OSC and 10 nonneoplastic peritoneal effusions. miR-21 was also evaluated by in situ hybridization.
Immunohistochemistry demonstrated a gradual PDCD4 loss from normal ovaries to CA and OSC specimens. Quantitative reverse transcriptase-polymerase chain reaction displayed higher PDCD4 messenger RNA levels in CA specimens compared with OSC cases and highlighted miR-21 overexpression in OSC specimens. In situ hybridization detected miR-21 only in OSC cells. This PDCD4 and miR-21 inverse expression was also noted in cells and exosomes from OSC peritoneal effusions compared with nonneoplastic effusions.
Ovarian cancer is the leading cause of gynecologic tumor-related death in the Western world.[1, 2] Malignant surface epithelial-stromal tumors account for approximately 90% of ovarian cancer cases, among which borderline and invasive ovarian serous carcinomas (OSC) represent the large majority. The majority of patients with OSC are initially asymptomatic and present with advanced stage disease (International Federation of Gynecology and Obstetrics stages III-IV), often characterized by bilateral localization and malignant peritoneal effusion.[2-6] Despite advances in treatment, survival is still low. Understanding the molecular alterations underlying OSC could provide the basis for novel diagnostic or therapeutic strategies.[3-6]
Programmed cell death 4 (PDCD4) is a recently discovered tumor suppressor gene involved in apoptosis that affects cell transformation, oncogenesis, and tumor invasion.[7-10] Function and subcellular localization of PDCD4 are controlled by several mechanisms, in particular by the oncogenic microRNA-21 (miR-21). Indeed, miR-21 directly targets the 3′-untranslated region of PDCD4, downregulating its expression.[11, 12] Moreover, low PDCD4 protein levels have been found to be inversely correlated with miR-21 expression and to correlate with prognosis in different tumors, such as thyroid, colon, esophageal, and ovarian carcinomas.[8, 11-15]
Exosomes are small (30–120 nanometers [nm] in size) membrane-bounded vesicles of cellular derivation detected in several bodily fluids, such as plasma, urine, saliva, breast milk, and effusions.[16-18] Exosomes have been demonstrated to participate in intercellular communication, delivering their cargo of molecules to specific target cells.[16-18] The transfer of molecules via exosomes may modulate the activity of cellular mechanisms and pathways in recipient cells. In particular, exosomes have been involved in oncogenesis, immunomodulation, angiogenesis, and metastasis.[16-18] Exosome content (proteins and RNA) is related to the cells of derivation. Indeed, exosomes are produced by a variety of cells, including lymphocytes and epithelial, endothelial, dendritic, and cancer cells.[16-18]
The objective of the current study was to assess miR-21/PDCD4 involvement in resected OSC specimens and in cells and exosomes from OSC peritoneal effusions.
MATERIALS AND METHODS
From the archives of the Surgical Pathology and Cytopathology Unit of the University of Padua for the period between 2009 and 2013, we retrieved resected specimens of 14 normal ovaries from cases of uterine prolapse, 14 serous cystadenoma (CA), and 14 OSC (clinical and pathological data for these cases are reported in Table 1). All cases were reviewed and the diagnoses confirmed in all instances by 2 pathologists (S.C. and R.C.) according to the World Health Organization classification.
Table 1. Clinicopathological Characteristics of the Considered Series
No. of Cases
Mean Age ± SD, Years
Histologic Grade, No.
FIGO Stage, No.
Bilaterality, No. (%)
Malignant Effusion, No. (%)
Abbreviations: CA, cystadenoma; FIGO, International Federation of Gynecology and Obstetrics; NEG, nonneoplastic; OSC, ovarian serous carcinomas; SD, standard deviation.
2 (3) 3 (11)
II (4) III (10)
2 (1) 3 (9)
II (2) III (8)
Peritoneal effusions were submitted for routine diagnostic purposes to the study unit and immediately processed. Fresh peritoneal effusions were collected between 2012 and 2013 from 10 patients diagnosed with OSC before the administration of chemotherapy and surgical treatment. Clinical and pathological data are detailed in Table 1. All effusions were proven to be malignant by cytology and OSCs were subsequently histologically confirmed in all instances. As a negative control, 10 nonneoplastic (cytologically negative) peritoneal effusions were also included.
This study was approved by the Institutional Ethical Review Board of Padua University and the institute's ethical regulations on research conducted on human tissues were followed.
Isolation of Cells and Exosomes
On their arrival at the laboratory, all effusion samples were divided in half: one half was used for the usual diagnostic process and the other for the study. The latter half was centrifuged for 6 minutes at 6000 g to separate the cells from the supernatant fluid. The cell pellet was readily stored at −80°C until further use, whereas the supernatant fluid was centrifuged again at 2000 g for 30 minutes to remove cellular debris and contaminants and 1 mL of the clarified fluid was transferred to a fresh container for isolation of the exosomes without disturbing the pellet. Then, 500 μL of Total Exosome Isolation Reagent (Invitrogen, Carlsbad, Calif), a polymer-based mixture that separates exosomes from solution by tying up water molecules thereby allowing their collection by low-speed centrifugation, were added and mixed by vortexing, according to the manufacturer's instructions. After incubation at room temperature for 30 minutes, the sample/reagent mixture was centrifuged at 10,000 g for 10 minutes at room temperature and the supernatant fluid was discarded. Finally, the exosome pellet was resuspended in 200 μL of 1X phosphate-buffered saline buffer and stored at −80°C until further use.
Immunohistochemistry was performed on 4-μm to 5-μm thick formalin-fixed and paraffin-embedded (FFPE) sections from each tissue sample. Staining was performed automatically (BondmaX; Menarini, Florence, Italy) as described elsewhere,[20, 21] using the Bond Polymer Refine Detection kit (Leica Microsystems, Wetzlar, Germany), with anti-PDCD4 antibody (polyclonal; working dilution, 1:100 for 30 minutes, citrate buffer) (Atlas Antibodies, Stockholm, Sweden). Sections were then slightly counterstained with hematoxylin. Appropriate positive and negative controls were run concurrently. PDCD4 nuclear expression was jointly scored by 2 pathologists (A.F. and R.C.) who were unaware of any clinical information. PDCD4 nuclear staining was scored semiquantitatively on a 4-tiered scale on the basis of the percentage of positive cells, with 0 indicating no stain, 1 indicating 1% to 30% staining, 2 indicating 31% to 70% staining, and 3 indicating 71% to 100% staining. Cytoplasmic expression was evaluated on a 4-tired scale based on the staining intensity, in which 0 indicated absent staining, 1 indicated weak staining, 2 indicated moderate staining, and 3 indicated strong staining. According to scoring system of Mudduluru et al, nuclear and cytoplasmic scores were summed to obtain a final value: 1 to 2 indicated weak expression, 3 to 4 indicated moderate expression, and 5 to 6 indicated intense expression.
In Situ Hybridization
Reactions were performed on 4-μm to 5-μm thick FFPE sections from 5 CA and 5 OSC randomly selected cases using the GenPoint Catalyzed Signal Amplification System (DakoCytomation, Glostrup, Denmark) according to the manufacturer's protocol and applying the 5′– biotin-labeled miR-21 miRCURY LNA microRNA detection probe (Exiqon, Vedbaek, Denmark) or the scrambled negative control probe (U6; Exiqon) at a final concentration of 200 nM, as described elsewhere. The slides were finally counterstained with hematoxylin. Reactions were jointly assessed by 2 pathologists (A.F. and R.C.) and were considered positive when granular cytoplasmic staining was present.
CA and OSC samples were enriched in the neoplastic component by manual microdissection to ensure a higher neoplastic component. Briefly, 5 consecutive, unstained, 10-μm thick, FFPE sections of each specimen were scraped in a 1.5-mL tube using the hematoxylin and eosin-stained slide as a guide. Total RNA was extracted using the RecoverAll Total Nucleic Acid Isolation Kit (Ambion, Austin, Tex) as described elsewhere.[23, 24] Cell pellets from peritoneal effusions were resuspended in 500 μL of TRIzol reagent (Invitrogen) for total RNA isolation, according to the manufacturer's instructions. RNA recovery from the exosome samples was performed with the Total Exosome RNA and Protein Isolation Kit (Invitrogen), following the manufacturer's protocol. Each sample was mixed with 200 μL of 2× denaturing solution, vortexed, and incubated on ice for 5 minutes. Then, 400 μL of acid-phenol:chloroform were added. The mixture was briefly vortexed and centrifuged at 10,000 g for 5 minutes at room temperature to separate the aqueous and organic phases. The aqueous phase was transferred to a fresh tube and a 1.25-volume of 100% ethanol was added. The mixture was then pipetted onto a filter cartridge in a collection tube and spun at 10,000 g for 15 seconds. The flow-through was discarded and the filter cartridge was washed once with 700 μL of Wash Solution 1 and twice with 500 μL of Wash Solution 2/3. After discarding the flow-through from the last wash, the filter cartridge was transferred into a fresh collection tube and 50 μL of preheated (95°C) nuclease-free water was twice applied to the center of the filter and briefly centrifuged to collect RNA. All RNA extractions were assessed for the amount and purity of RNA with Nanodrop technology (Thermo Scientific, Wilmington, Del) and stored at −80°C until further use. To avoid any potential variation between assays, analyses were then performed on all of the extracts simultaneously.
PDCD4 reverse transcription was performed using 100 ng of total RNA, M-MLV Reverse Transcriptase (Invitrogen), and 250 mM of random primers (Invitrogen). PDCD4 primers (forward 5′-TGGAAAGCGTA AAGATAGTGTGTG-3′; reverse 5′-TTCTTTCAGCA GCATATCAATCTC-3′) were designed using the ProbeFinder software (roche-appliedscience.com) and the respective probe was selected among the Universal ProbeLibrary (Roche Diagnostics, Mannheim, Germany). Experiments were performed according to the standard protocol provided by the manufacturer, including a housekeeping gene control (β-2 microglobulin) to adjust for unequal RNA amounts, as explained elsewhere. Mature miR-21 (primer sequence 5′-GATACCAAAAT GTCAGACAGCC-3′) was retrotranscribed from 100 ng of total RNA with the SuperScript VILO cDNA Synthesis Kit (Invitrogen) and quantified using the NCode miRNA quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) method (Invitrogen), according to the manufacturer's instructions, as previously stated.[25, 26] Normalization was done with the small nuclear RNA U6B (RNU6B; Invitrogen). All the reactions were run in triplicate, including no-template controls, on the LightCycler 480 Real-Time PCR System (Roche Diagnostics).
Statistical significance was determined for immunohistochemistry results using the Kruskal-Wallis test. Differential expression of PDCD4 and miR-21 was tested on the logarithmic scale using a 2-sided Student t test, after checking both the assumption of normality (Shapiro-Wilk test) and the assumption of homogeneity of variance (F-test). In situ hybridization results were not statistically analyzed. A P value < .05 was considered to be statistically significant. All statistical analyses were performed using R software (R Development Core Team, version 2.9; R Foundation for Statistical Computing, Vienna, Austria).
In normal ovaries, PDCD4 immunostaining demonstrated a moderate cytoplasmic intensity and a strong nuclear positivity in virtually all surface cells (Fig. 1), with an intense final score in all instances (Fig. 2). CA specimens displayed a moderate-to-strong cytoplasmic immunoreaction with a scattered nuclear positivity (Fig. 1). Overall, PDCD4 immunolabeling scoring resulted an intense score in 21%, a moderate score in 71%, and a weak score in 8% of CA specimens (Fig. 2). In OSC specimens, nuclear immunoreaction was found to disappear completely whereas cytoplasmic immunostaining ranged in intensity from weak to moderate (Fig. 1). Approximately one-half of OSC cases displayed a weak final expression whereas the remaining specimens were found to have a moderate score (Fig. 2). Statistical analysis of immunohistochemistry results highlighted a substantial decrease in PDCD4 expression in both OSC and CA specimens compared with normal ovary (all P < .001). Such findings were further supported by qRT-PCR analysis of PDCD4 expression in CA and OSC specimens. Indeed, PDCD4 messenger RNA (mRNA) levels were found to be significantly higher in CA specimens compared with OSC specimens (P < .05) (Fig. 2). Conversely, miR-21 was found to be overexpressed in OSC compared with CA specimens (P < .05) (Fig. 2). This finding was also confirmed by in situ hybridization analysis performed in a limited number of CA and OSC cases that demonstrated considerable granular cytoplasmic staining in OSC cells only (Fig. 3).
Inverse expression of PDCD4 and miR-21 also were confirmed in cells and exosomes from OSC peritoneal effusions compared with nonneoplastic effusions. Indeed, qRT-PCR results demonstrated higher PDCD4 mRNA levels in the cells and exosomes of nontumoral compared with OSC effusions (both P < .01) (Table 2) (Fig. 4). Conversely, miR-21 was found to be significantly downregulated in both cells (P < .05) and exosomes (P < .01) from nonneoplastic controls in comparison with OSC effusions (Table 2) (Fig. 4). Thus, exosomes in OSC peritoneal effusions presented with high miR-21 content similar to that of the presumed cells of origin (ie, OSC cells).
Table 2. qRT-PCR Results in Cells and Exosomes Obtained From OSC and NEG Peritoneal Effusions
Ovarian cancer is the leading cause of gynecologic tumor-related deaths in the Western world and OSC represents the most common histologic subtype.[1, 2] Early symptoms are usually absent, minor, and overlooked, and the large majority of patients present with advanced disease.[2-6] This is the main reason for the poor prognosis of OSC and there is a pressing need for the development of novel diagnostic and therapeutic strategies.[3-6]
The current study investigated the expression of the recently discovered tumor suppressor gene PDCD4 and its regulator miR-21 among a series of normal ovary, CA, and OSC surgical specimens and in cells and exosomes from OSC peritoneal effusions. PDCD4 is a tumor suppressor gene located at chromosome 10q24 that is associated with apoptosis-regulating activator protein 1 (AP-1)-dependent transcriptional activity, cyclin-dependent kinase 4 (CDK4), p27, p21, and eukaryotic translation initiation factor 4A (eIF4A) in response to several inducers.[7-10] PDCD4 loss or downregulation has been correlated with tumor progression and poor prognosis in different tumors, such as thyroid, colon, esophageal, and ovarian cancers.[8, 11-15] In ovarian cancer cell lines, PDCD4 overexpression has been demonstrated to inhibit the malignant behavior enhancing apoptosis and chemosensitivity.[27-30] The results of the current study in resected samples are in keeping with data from the literature. Indeed, a substantial decrease in PDCD4 levels was observed in OSC specimens compared with normal controls and CA specimens at both the protein and mRNA level, as reported by other authors.[14, 15]
Previous studies have demonstrated that the function and subcellular localization of PDCD4 are controlled by several mechanisms, mainly miR-21.[11-13] Indeed, miR-21 is an oncogenic microRNA that recognizes and directly binds the 3′-untranslated region of PDCD4, thereby controlling its expression. Nam et al found overexpression of miR-21 in OSC specimens. Moreover, miR-21 downregulation has been reported to promote apoptosis and chemosensitivity, while conversely inhibiting migration and invasion in ovarian cancer cell lines.[29, 31, 32] Accordingly, the results of our qRT-PCR demonstrated higher expression of miR-21 in OSC compared with CA cases, findings that were further confirmed by in situ hybridization. Overall, such findings endorse the involvement of PDCD4/miR-21 in the oncogenesis of OSC.
It is interesting to note that the same inverse expression of PDCD4 and miR-21 was maintained also in cells and exosomes from OSC peritoneal effusions compared with nonneoplastic effusions. These characteristics reveal that the frequent bilaterality and multiple peritoneal localization of OSC could be based on double mechanisms. The first and more obvious mechanism is direct adhesion and proliferation of neoplastic cells to other sites of the peritoneal cavity, a point that does not need further comments. The latter and intriguing process is unrelated to the direct migration of OSC cells and depends on exosomes. Indeed, exosomes could promote oncogenic transformation in target cells distant from the primary tumor. These 30-nm to 120-nm sized vesicles play a role in intercellular communication by transferring proteins and RNA from donor to recipient cells.[16-18] Exosomes are implicated in several tumor mechanisms, such as oncogenesis, immunomodulation, angiogenesis, and metastasis.[16-18] In particular, many of these functions are related to the transfer of miRNAs in recipient cells, in which they modulate tumor transformation, proliferation, and progression.[16-18, 33, 34] miRNAs are selectively incorporated into exosomes and delivered to specific cells, although it remains unclear whether the sorting mechanism is related to the association with the RNA-induced silencing complex (RISC) components or the target mRNAs.[16, 35, 36] Moreover, the detection of miR-21 in exosomes also could be used as a diagnostic tool in peritoneal effusions. Recently, Taylor et al demonstrated that miR-21 levels were higher in exosomes isolated from the sera of patients with OSC than those from women with benign disease and precancerous lesions. In addition, miR-21 was included in a panel of miRNAs from tumor-derived exosomes whose expression analysis was effective for diagnosing OSC. Currently, exosome analysis is performed only for research purposes. However, the availability of fast, simple, and inexpensive isolation methods could soon increase its application as an ancillary technique to effusion cytology (and it most likely will replace light microscopy evaluation in the future). Currently, exosome analysis is 5 to 6 times more costly than routine cytology, thereby limiting its diffusion.
The results of the current study demonstrate that the loss of PDCD4 with a concurrent increase in miR-21 expression occurs in OSC, confirming their involvement in the oncogenesis of the neoplasm. The presence of miR-21 in OSC exosomes suggests the involvement of this mechanism in the oncogenic transformation of target cells distant from the primary tumor. This could be an alternative to direct colonization by cancer cells underlying the multiple peritoneal localizations commonly observed in OSC. In addition, the presence of high miR-21 expression levels in the cells and exosomes of OSC peritoneal effusions could be used as a diagnostic tool.