s2fieldsofstudy: Biology Medicine

text:
PD-L1 and immune infiltrates are differentially expressed in distinct subgroups of gastric cancer

ABSTRACT This study investigates the association of PD-L1 expression and immune cell infiltrates and their impact on clinical outcome, in addition to their overlap with microsatellite instability (MSI), HER2 and ATM molecular subgroups of gastric cancer (GC). PD-L1 membrane expression on tumour cells (TC) and infiltrating immune cells (IC), CD3 + T-lymphocytes, CD8+ cytotoxic T-cells, ATM and HER2 were assessed by immunohistochemistry (IHC) in the ACRG (Asian Cancer Research Group) GC cohort (N = 380). EBV status was determined using in situ hybridization and MSI status was performed using PCR and MLH1 IHC. The PD-L1 segment was associated with increased T-cell infiltrates, while the MSI-high segment was enriched for PD-L1, CD3, and CD8. Multivariate analysis confirmed PD-L1 positivity, high CD3 and high CD8 as independent prognostic factors for both disease-free survival and overall survival (all p < 0.05). Patients with MSI-high tumours had better overall survival by both univariate and multivariate analysis. The ATM-low and HER2-high subgroups differed markedly in their immune profile; the ATM-low subgroups enriched for MSI, PD-L1 positivity and CD8 + T-cells, while the HER2 segment was enriched for MSS, with no enrichment for immune markers. Hence, we demonstrate a molecular profiling approach that can divide GC into four molecular subgroups, namely ATM-low, HER2-high, PD-L1 positive and MSI-high with differing levels of immune infiltrates and prognostic significance which may help to stratify patients for response to targeted therapies.

Introduction
Gastric cancer (GC) is the fifth most common malignancy worldwide, the third leading cause of cancer mortality, and occurs with a high incidence in East Asia. 1 GC is often diagnosed at an advanced stage, for which therapeutic options are largely limited to cytotoxic chemotherapy and five-year survival is less than 20%. 2 Despite the availability of Trastuzumab for use in human epidermal growth factor receptor 2 (HER2)-positive disease and the anti-VEGFR2 antibody ramucirumab for use as a second line therapy, prognosis remains poor, with an urgent need for effective targeted therapies and an understanding of those patients most likely to benefit. [2][3][4] The interaction of programmed cell death-1 (PD-1) on T-cells with its ligand, programmed cell death ligand-1 (PD-L1) on immune and tumour cells, limits T-cell mediated responses. 3 Immune checkpoint blockade with anti-PD1 or anti-PD-L1 antibodies is emerging as a promising therapeutic approach for several cancer types, including non-small cell lung cancer (NSCLC), [4][5][6] melanoma, 7 bladder 8 and renal cancer. 9 In early clinical studies anti-PD-1 therapies, including pembrolizumab 10 have reported promising efficacy in metastatic GC and recently, a phase III trial comparing nivolumab versus best supportive care in the salvage setting demonstrated survival benefit in GC. 11 Tumour PD-L1 expression is being explored as a predictive biomarker for response to these agents, 10,12 however not all PD-L1 positive patients respond and responses occur in patients with PD-L1 negative tumours, hence alternative markers are required. Microsatellite instability (MSI) predicts response to PD-1/PD-L1 blockade, 13 potentially related to the high mutational burden and neoantigen generation associated with MSI-high tumours 14 and approximately 22% gastric tumours are MSI-H. 13,15 Another pathway, with the potential to modulate the response to immune checkpoint inhibition, is the cellular DNA damage repair pathway, which is necessary to maintain genome stability and in which the ataxia-telangiectasia-mutated (ATM) kinase plays an essential role. 16 Loss of function variants in ATM confer an increased risk of GC 15 and approximately 10% to 20% of GCs have low or undetectable ATM expression as assessed by immunohistochemistry (IHC) and this subtype is enriched with MSI. 17,18 Based on the association with the cellular DNA damage response and mismatch repair, we hypothesized that the ATM low could be an immunologically primed subtype.
Here we have assessed the association of PD-L1 expression and immune cell infiltrates with clinicopathological features and outcome and investigated the overlap of these with MSI-high, ATM low and HER2-high segments. In addition, we have correlated the peritumoral immune cell infiltrates according to gene expression based molecular classifications in GC. 19

Patient and tumour characteristics
We procured 380 primary GC specimens at the time of total or subtotal gastrectomy from Samsung Medical Center. We selected the cases based on > 60% histological purity and sufficient tumor blocks for additional immunohistochemistry (IHC). Of 380 specimens, 300 samples were previously profiled with gene expression signatures. 19 The remaining 80 specimens were not qualified for gene expression profiling but qualified for further IHC staining, thus included in this study (Sup. Figure 1). Patient and tumour characteristics of this Korean GC cohort (n = 380) are summarized in Table 1. Of 380 specimens, there was a good balance of tumours representative of the two major Lauren subgroups, with 45.5% (n = 173) intestinal and 51.1% (n = 194) diffuse type and the remainder comprised of 2.9% mixed subtype and 0.5% indeterminate subgroups. The frequency of EBV positive tumours was 6.6% (n = 25). Tumours with high HER2 expression assessed by immunohistochemistry (IHC 3+) occurred with a frequency of 7.2% (n = 27).

Prevalence of PD-L1, CD3 and CD8 T-cells in GC
PD-L1 expression on both the tumour cell (TC) membrane compartment and infiltrating immune cells (IC) was assessed by IHC analysis and PD-L1 positivity was defined as ≥ 1% of cells staining positive. This showed that 16.4% of tumours (62/379) scored positive for PD-L1 TC expression, while 90.2% (339/376) were positive for PD-L1 IC expression (Table 2). CD3 expression was assessed as a marker of global T-lymphocytes and CD8 expression as a marker of cytotoxic T-cells. Cut-offs for defining CD3 and CD8 high versus low were identified by investigating the associations between outcome and CD3 or CD8 positive cell count, where the cut-off giving the largest significant survival hazard ratio was selected (in silico validation Sup. Figure 2). Using these cut-offs of 500 cells/mm 2 to define low versus high CD3 and 600 cells/ mm 2 for low versus high CD8 expression, high CD3 expression was observed in 69.7% (242/347) tumours, while high CD8 expression was observed in 25.6% (89/347) of tumours ( Table 2). A positive correlation was observed between CD3 high and CD8 high tumours (p < 0.01; data not shown). Increased TC or IC PD-L1 staining were significantly associated with increased CD3+ lymphocyte infiltration (PD-L1 TC Spearman correlation 0.34, p < 0.01; PD-L1 IC Spearman correlation 0.37, p < 0.01) and with CD8+ cytotoxic T-cell density (PD-L1 TC Spearman correlation 0.41; p < 0.01; PD-L1 IC Spearman correlation 0.42, p < 0.01).
Association of PD-L1, CD3 and CD8 expression with clinicopathological features Tumours with MSI were associated with increased TC and IC PD-L1 staining (Figure 1(a,c): PD-L1 TC P < 0.01; PD-L1 IC P < 0.001) and with increased CD3 and CD8 immune cell densities (Figure 1(a,c): CD3 P < 0.05; CD8 P < 0.01). Statistical analysis indicated that, whilst there was no evidence for a significant association between Lauren subtype and PD-L1 TC or CD8, there was a significant association between Lauren subtype and PD-L1 IC and CD3, where diffuse type GC had significantly lower PD-L1 IC expression (p < 0.01) and significantly higher CD3 cell infiltrates (p = 0.027), compared to intestinal type GC ( Figure 1d). Of note, EBV+ GC demonstrated higher percentage of PD-L1 positivity on tumour and immune cells when compared to non-EBV GC tumours (p < 0.001, Figure 1e) and also had a higher incidence of both CD3 and CD8 positive immune infiltrates (p < 0.01, Figure 1e).

Association of PD-L1, CD8 and CD3 immune markers with survival
The median disease-free survival (DFS) and overall survival (OS) in this patient cohort were 57.2 (38.9-75.4) and 64.0 (47.7-77.2) months, respectively (95% confidence intervals). Both DFS and OS were significantly longer in GC patients with high CD3 (p < 0.01) or CD8 (p < 0.01) immune infiltrates, compared with patients with low CD3 or CD8 GCs (Figure 2(c,d)). DFS and OS were significantly improved for patients with positive (≥ 1%) TC PD-L1 expression (OS p < 0.01) compared to negative TC PD-L1 expression ( Figure 2a). In contrast, PD-L1 IC positivity was not associated with survival ( Figure 2b). However, when the cut-off for defining PD-L1 IC positivity was raised to ≥ 5%, a significant association with both DFS and OS was observed (P < 0.01, Sup. Figure 3). Using this higher cut-off, 56% (212/379) tumours were PD-L1 IC positive (Table 2). Multivariate analysis, adjusting for age, gender, Lauren subtype, TNM stage and metastasis, confirmed positive PD-L1 tumour membrane status (P < 0.01) and both high CD3 (OS P < 0.01; DFS p = 0.021) and high CD8 (OS P < 0.01; DFS p = 0.027) immune infiltrates as independent prognostic factors to predict better OS and DFS following surgery (Table 3).  As we previously reported, 19 patients with MSI-high tumours had better overall survival by both univariate and multivariate analysis (Table 3, p < 0.01 univariate and P < 0.05 multivariate). Moreover, when we divided the MSI or MSS cohort into sub-groups of either PD-L1 TC positive or negative tumours, within each cohort PD-L1 positive tumours had an improved OS (MSI p = 0.031; MSS p = 0.039), with the best outcome observed for patients with MSI-high/PD-L1 positive tumours and those with MSS/PD-L1 negative tumours having the worst outcome (Figure 1b). Hence, MSI status and PD-L1 status in TC combine to influence the outcome of GC patients.

Discussion
Here we have explored the association of the immune markers PD-L1, CD3 and CD8 with outcome and with other marker defined subgroups, namely ATM, HER2 and MSI, in a large Asian GC cohort. We show that TC PD-L1 expression is an independent prognostic factor predictive for better outcome in Asian GC patients. Furthermore, TC PD-L1 correlates with increased T-lymphocytes and cytotoxic T-cell infiltrates, as assessed by CD3 and CD8 marker expression and these markers are also prognostic for improved survival. PD-L1 status has been correlated with both favorable and unfavorable outcome in different cancers 20-26 and increased immune prevalence has also been reported to associate with improved clinical outcome. 27,28 Previous GC studies have been contradictory, reporting that PD-L1 is associated with both good 29-32 and poor prognosis [33][34][35] or has no association with survival. 36 The reason for these contradictory findings are uncertain, but could be attributable to use of different PD-L1 antibodies, assays and scoring systems or alternatively represent differences between GC cohorts. Overall this study shows that in a Korean cohort of patients with immuno-competent gastric tumours have a better outcome and supports exploration of blockade of the PD-1/PD-L1 checkpoint as a potential therapeutic strategy for GC. Furthermore, PD-L1 staining and CD3/CD8 immune infiltrates are significantly associated with MSI-high and these observations are consistent with reports in GC 37 and CRC 14 and clinical data has shown that mismatch repair deficiency predicts for response to PD1 blockade. 13 Our data are also in agreement with several recent studies that demonstrated an association between PD-L1 and EBV-associated malignancies. 37,38 Overall our findings suggest that EBV and MSI-high tumours are well placed to benefit from immune therapy. In contrast, the lack of association between immune markers and Lauren subtype suggests that patient stratification by Lauren classification alone may not be sufficient for PD-L1 therapy selection in immuno-competent GC. The cut-off value for PD-L1 positivity and comparison to other available PD-L1 antibodies for IHC should be evaluated in future studies.
Trastuzumab is the only targeted therapy currently approved for GC and high tumour HER2 expression by IHC is used to select patients for this therapy. 2,39,40 Another molecular subtype of GC, identifiable by IHC analysis, are ATM low expressing tumours but no approved targeted therapy exists for this molecular subtype. In the current study, it is clear that the ATM low and HER2 high segments are largely mutually exclusive with contrasting immune profiles. The ATM low segment is enriched for PD-L1, CD8 positive cytotoxic T-cells and MSI-high status, while the majority of HER2 high tumours are MSS with no enrichment for PD-L1 positivity nor for CD8 immune infiltrates. This suggests that the ATM-low segment is well placed to benefit from anti-PD-L1/ PD1 therapy. Interestingly within the subset of ATM positive tumours those cancers with the highest level of ATM expression (IHC 3+) had a worse outcome, together with a lower prevalence of both MSI-high and PD-L1 expression. ATM plays an essential role in the cellular DNA damage response   necessary to maintain genome stability and clinically ATM mutations are associated with microsatellite mutations in the ATM gene. 18,41,42 The genomic instability induced by low/loss of ATM expression could result in the generation of neoantigens and an immunologically "hot" tumour microenvironment. This ATM low segment could benefit from combination therapy with immunotherapy and DDR inhibitors. Although in a Phase III study, the PARP inhibitor olaparib failed to deliver the expected benefit (Bang et al., ESMO 2016), other DDR inhibitors such as ATR inhibitors should be tested in GC, especially in those tumours that are ATM-low and PD-L1 positive. In contrast, it could be argued that the HER2 high segment is less likely to benefit from PD-L1/PD1 checkpoint therapy alone. In this regard it will be of interest to understand the effects of trastuzumab therapy on the tumour immune environment.
Here we have assessed the overlap and immune marker expression of GCs divided into four distinct molecular subgroups, namely ATM-low, HER2-high, PD-L1 positive and MSI-high ( Figure 5). Significant overlap exists between the ATM low, PD-L1 and MSI-high subgroups. Notably the ATM low and HER2 high segments are mutually exclusive and differ markedly in their immune profile, the "immunologically hot" ATM low segment being enriched for MSI-high, PD-L1 and CD8 while the HER2 segment is enriched for MSS, with no enrichment for immune markers. The PD-L1 segment is associated with T-cell infiltrates as assessed by CD3 and CD8 expression and is prognostic by multivariate analysis for better outcome, while the MSI-high segment is enriched for immune markers including PD-L1 and has significant overlap with the ATM-low but not HER2 segments. Recently pembrolizumab was approved by the U.S. FDA for use in any solid tumour with MSI-high (https://www.fda.gov/newsevents/ newsroom/pressannouncements/ucm560167.htm). In our study, when overlaid with the known ACRG molecular subgroups, PD-L1 was highly enriched in MSI subtype.
Limitations of this study include that it is a retrospective analysis conducted on a GC cohort collected at a single Korean institution. While these observations do need to be confirmed in Western GC, it has similarly been reported that PDL1 is associated with EBV+, MSI-high and is an independent survival prognosticator in a Western cohort of 465 GC (Boger et al). In the current study tumour samples are from patients undergoing primary resection and further studies are required to determine whether the observations apply to metastatic cancers.
In conclusion, this study adds to the body of evidence illustrating that in GC PDL1, CD3/CD8 and MSI-high are prognostic markers and highlights the potential for molecular subsets of GC to respond differently to immune therapy.

Materials and methods
For full details please see online supplementary materials and methods

Cohort
Samsung Medical Centre, Seoul, Korea, procured the n = 380 primary independent GC specimens at the time of total or subtotal gastrectomy from 2004-2007. 19 Of the initial 380 patients, 347 met the full study inclusion criteria (Sup. Fig. 1). All of the tissue specimens were at chemo-naïve state during primary resection of gastric cancer. Samsung Medical Centre Institutional Review Board (IRB no. 2010-12-088) approved the protocol and informed consent was obtained according to the IRB protocol.

Ebv-encoded RNA in situ hybridization
The in-situ hybridization procedure was performed on the fully automated BOND-MAX system, with an EBV-encoded RNA probe (Leica Biosystem) following the manufacturer's instructions. Only cases with a strong signal within more than 95% of tumour cell nuclei were considered to be positive.

Microsatellite instability
The MSI test was performed using multiplex PCR comprising five quasimonomorphic mononucleotide repeat markers (NR27, NR21, NR24, BAT25, and BAT26). 45 Samples with no allelic size variations in any of the microsatellites were classified as microsatellite stable (MSS). Tumours with allelic size variations in one (NR24) or more of the microsatellites were considered MSI-high.

Statistical analysis
Associations between categorical variables were assessed for significance by Fisher's exact test. Associations between numerical and categorical markers were assessed by Kruskal-Wallis test and\or Mann-whitney test. Median survival times and confidence intervals were calculated by Kaplan Meier estimation. Hazard ratios were calculated by Cox proportional hazards modelling, with each marker modelled by a univariate model and also by a multivariate model incorporating age, gender, Lauren subtype, TNM stage and metastasis as covariates. For all statistical analysis p < 0.05 was considered statistically significant. Differences in survival were also considered significant at p < 0.05. No consideration was given to multiplicity of testing due to the exploratory nature of this study. In silico validation of the CD3 and CD8 data were achieved, where optimal cut-points were determined using the k-folds validation approach with bootstrapping. Agreement was seen with those determined from a visual assessment of hazard ratio and associated confidence intervals (Sup. Figure 2).


extfieldsofstudy: Chemistry Medicine

text:
Visualization of Annular Gap Junction Vesicle Processing: The Interplay Between Annular Gap Junctions and Mitochondria

It is becoming clear that in addition to gap junctions playing a role in cell–cell communication, gap junction proteins (connexins) located in cytoplasmic compartments may have other important functions. Mitochondrial connexin 43 (Cx43) is increased after ischemic preconditioning and has been suggested to play a protective role in the heart. How Cx43 traffics to the mitochondria and the interactions of mitochondria with other Cx43-containing structures are unclear. In this study, immunocytochemical, super-resolution, and transmission electron microscopy were used to detect cytoplasmic Cx43-containing structures and to demonstrate their interactions with other cytoplasmic organelles. The most prominent cytoplasmic Cx43-containing structures—annular gap junctions—were demonstrated to form intimate associations with lysosomes as well as with mitochondria. Surprisingly, the frequency of associations between mitochondria and annular gap junctions was greater than that between lysosomes and annular gap junctions. The benefits of annular gap junction/mitochondrial associations are not known. However, it is tempting to suggest, among other possibilities, that the contact between annular gap junction vesicles and mitochondria facilitates Cx43 delivery to the mitochondria. Furthermore, it points to the need for investigating annular gap junctions as more than only vesicles destined for degradation.

Introduction
Gap junction channels play a pivotal role in a vast number of physiological events by providing channels for the intercellular communication of regulatory molecules between cells [1]. Gap junction channels are composed of gap junction proteins, termed connexins [2]. All of the members of the multigene connexin family have a similar topology and the sequences of several connexin gap junction proteins have been determined [3,4]. Twenty-one different connexins have been identified in humans [5]. Connexin 43 (Cx43) gap junction protein, the most ubiquitously expressed connexin, is thought to be synthesized in the endoplasmic reticulum, oligomerized into a hemichannel in the Golgi [6] and then transported to the cell surface in secretory vesicles. On the cell surface, hemichannels from apposing cells align (dock) to form channels [7]. Gap junction channels then aggregate to form gap junction plaques [1]. These gap junction plaques are composed of thousands of channels [6] and plaque size is determined by the number of hemichannels that are delivered to the membrane in secretory vesicles and aggregated on the surface to form these plaques [7].
Gap junction plaques are removed from the cell surface by an internalization event [8,9]. The disassembly and removal of gap junction plaques from the plasma membrane involve a clathrin/dynamin-dependent unique internalization process [10][11][12] in which the gap junction plaque that connects two adjacent cells is internalized into the cytoplasm of one of the two cells to form an annular gap junction [13][14][15][16]. These annular gap junctions were first identified with transmission electron microscopy (TEM) and were distinguished from other organelles by the presence of a double-membrane (pentalaminar membrane) and a central lumen [8,[13][14][15][16][17][18]. Annular gap junctions (also called connexosomes) have now been demonstrated with live cell imaging techniques to form from internalization at central regions of the gap junction plaque [19][20][21][22] or internalization of the entire gap junction plaque [23].
The central dogma has been that the gap junction proteins (connexins) in these annular gap junctions are degraded by autophagosomal and endo/lysosomal pathways [24][25][26][27][28][29], and thus the internalization process is only a method of eliminating old connexins [8,15,30]. However, data from our laboratory and that of others has demonstrated that connexins, specifically annular gap junction vesicle connexin 43 (Cx43), can recycle back to the cell surface to form functional gap junctions [31,32]. While the aggregation of gap junction channels into plaques and channel gating have been extensively studied [19,27,28], mechanisms involved in the processing of annular gap junction vesicles, and the organelles they interact with, have only recently gained attention. However, it is clear that the rates of gap junction plaque disassembly as well as organelle interactions are critical to gap junction function in cell-cell communication and cell-cell adhesion [29,30]. Further, changes in gap junction location, connexin trafficking, and internalization is thought to play several pivotal physiological roles during embryonic development [33,34], mitosis [32], wound healing [35,36], and cell migration [35]. Furthermore, gap junction plaque changes are thought to facilitate tumor growth and metastasis [34] as well as play a pivotal role in the cardiac electrophysiology and the survival of cardiomyocyte during ischemia [37]. Additionally, Cx43, located in mitochondria, is thought to regulate mitochondrial function (oxygen consumption and potassium fluxes) [38]. Mitochondrial Cx43 is altered under several pathophysiological cardiac conditions including, hypertension, hypertrophy, hypercholesterolemia, ischemia/reperfusion injury, post-infarction remodeling, and heart failure [38][39][40][41][42]. The possible pathophysiological role of Cx43 within cytoplasmic compartments or associations between these compartments with mitochondria is not known. Understanding Cx43 trafficking, fate and the translocation to Cx43-containing organelles is essential to an understanding of a host of normal and pathological events.
In this study, we demonstrated cytoplasmic Cx43-containing structures and evaluated their organelle interactions. We found that the most abundant Cx43-containing structures, annular gap junction vesicles, associated more frequently with mitochondria than with lysosomes. These findings are consistent with the possibility that annular gap junction and mitochondria may communicate with one another and that annular gap junction Cx43 may be utilized rather than being only degraded.

Characterization of Cx43-Containing Gap Junctions' Structure Behavior
A human adrenal tumor cell line (SW-13) that expresses Cx43 gap junction protein was used throughout this study to analyze cytoplasmic Cx43-containing structures, particularly that of annular gap junction vesicles, which are the most prominent Cx43-containing organelle in the cytoplasm ( Figure 1A). This cell line forms large gap junctions that are spontaneously internalized to form annular gap junction vesicles, which is particularly advantageous for studying aspects of Cx43 trafficking. With immunocytochemical labeling of cells expressing endogenous Cx43 and live cell imaging of cells expressing Cx43-GFP, gap junction plaques can be seen at the cell surface between contacting cells (Figures 1a,b and 2). Puncta indicative of annular gap junction vesicles could be easily seen within the cytoplasm (Figures 1 and 2). In addition, secretory vesicles that deliver new Cx43 to the plasma membrane could be detected at the light level of resolution, but they were more easily discerned with super-resolution microscopy ( Figure 1C). Secretory and annular gap junction vesicles both appeared as puncta at the light microscopic level of resolution and are distinguished from one another mainly by the differences in their sizes. Annular gap junction vesicles (≥0.5 µm) have been previously demonstrated to be much larger than secretory vesicles (≤150 nm) [19,40].
With transmission electron microscopy gap junction plaques as well as annular gap junction vesicles could be positively identified, depending on the plane of focus, by either the typical pentalaminar membrane ( Figure 1D,E, white arrow or arrow head) or the densely stained appearance of the double membrane ( Figure 1D,E, black arrow or arrow head). easily discerned with super-resolution microscopy ( Figure 1C). Secretory and annular gap junction vesicles both appeared as puncta at the light microscopic level of resolution and are distinguished from one another mainly by the differences in their sizes. Annular gap junction vesicles (≥0.5 µm) have been previously demonstrated to be much larger than secretory vesicles (≤150 nm) [19,40]. With transmission electron microscopy gap junction plaques as well as annular gap junction vesicles could be positively identified, depending on the plane of focus, by either the typical pentalaminar membrane ( Figure 1D,E, white arrow or arrow head) or the densely stained appearance of the double membrane ( Figure 1D,E, black arrow or arrow head). Immunocytochemistry of endogenous Cx43 (red) and cortical actin (green). Cortical actin (green) staining was used to define the boundaries of the cell and to aid in distinguishing intracellular annular gap junction puncta (red, arrow) from surface gap junction plaques (red, arrowhead). (B) Pseudocolored confocal microscopic image of Cx43-GFP from a Time-lapse frame with gap junction plaques (arrowhead) and annular gap junction vesicles (arrow) demonstrate the differences in Cx43-GFP intensities. (C) Pseudocolored super-resolution microscopic image demonstrating Cx43-GFP intensities of annular gap junction (arrow) and secretory vesicles (broken arrow). The Look Up Table  (LUT) scale in B and C indicates the intensities of GFP in the gap junction structures. (D,E) TEM of a gap junction plaque (D) and annular gap junction vesicles (E) identified, depending on the plane of focus, by typical pentalaminar membrane (white arrow head or arrow) or the appearance of the densely stained double membrane (black arrowhead and arrow). (F) Bizarre annular gap junction vesicle with annular structure and other unknown material within the lumen. The pentalaminar membrane (white arrows) and immuno-electron microscopic Cx-43 Q-dots labeling (black arrows) was used to identify the annular gap junction vesicle. Scale Bars = (A,B):10 µm, (C): 5 µm, (D-F): 100 nm. Immunocytochemistry of endogenous Cx43 (red) and cortical actin (green). Cortical actin (green) staining was used to define the boundaries of the cell and to aid in distinguishing intracellular annular gap junction puncta (red, arrow) from surface gap junction plaques (red, arrowhead). (B) Pseudocolored confocal microscopic image of Cx43-GFP from a Time-lapse frame with gap junction plaques (arrowhead) and annular gap junction vesicles (arrow) demonstrate the differences in Cx43-GFP intensities. (C) Pseudocolored super-resolution microscopic image demonstrating Cx43-GFP intensities of annular gap junction (arrow) and secretory vesicles (broken arrow). The Look Up Table ( In addition to annular gap junctions and secretory vesicles, ultrastructural analysis revealed atypical aggregates of gap junction membrane vesicles ( Figure 1F). These structures were identified by the typical gap junction membrane while the more bizarre structures were further confirmed as composed of Cx43 with Q dot immuno-electron microscopy ( Figure 1F). Such structures were very rare with an occurrence of less than 10 in 549 images of annular, 0.02%. Given the recent suggestion that Cx43 in annular gap junctions could have fates other than only degradation [31,32], we did a detailed analysis of annular gap junction processing and their associations with other organelles.  In addition to annular gap junctions and secretory vesicles, ultrastructural analysis revealed atypical aggregates of gap junction membrane vesicles ( Figure 1F). These structures were identified by the typical gap junction membrane while the more bizarre structures were further confirmed as composed of Cx43 with Q dot immuno-electron microscopy ( Figure 1F). Such structures were very rare with an occurrence of less than 10 in 549 images of annular, 0.02%. Given the recent suggestion that Cx43 in annular gap junctions could have fates other than only degradation [31,32], we did a detailed analysis of annular gap junction processing and their associations with other organelles.
2.1.1. Live Cell Imaging of Gap Junction Plaque Endoexocytosis in Cells Expressing Cx43-GFP Since at the light microscopic level distinguishing the pentalaminar or dense double membranes was not possible, positive identification of annular gap junctions in these live cell imaging studies was made by monitoring gap junction plaque internalization and thus following annular gap junctions observed to form in cells expressing Cx43-GFP. Annular gap junctions were observed to form both as a result of internalization of portions of gap junction plaques or entire gap junction plaques. In both cases the annular gap junction vesicle was released into one of two contacting cells ( Figure 2). We were unable to track or analyze the smaller gap junction vesicles since they quickly went out of focus. Of the annular gap junctions that were tracked, the average puncta size, measured from live cell imaging of Cx43-GFP expressing cells, was 1.6 ± 0.3 µm 2 , a size previously shown to be consistent with annular gap junctions and not secretory vesicles [19]. In Figure 2, a gap junction plaque is shown to first invaginate to produce a gap junction bud which in time detaches to form an annular gap junction vesicle. Over a 7 h viewing period 24.0 ± 3.4 annular gap junctions were observed to form.
As summarized in Figure 2H, over a 26 h monitoring period, 93 annular gap junctions, selected for tracking analysis, from four different movies, were observed to either (a) fragment to form smaller puncta, (b) join with cytoplasmic organelles, (c) disappear from view, or (d) move toward and appear to associate with the cell surface gap junction plaques. Note, while other puncta were observed to be Live Cell Imaging of Gap Junction Plaque Endoexocytosis in Cells Expressing Cx43-GFP Since at the light microscopic level distinguishing the pentalaminar or dense double membranes was not possible, positive identification of annular gap junctions in these live cell imaging studies was made by monitoring gap junction plaque internalization and thus following annular gap junctions observed to form in cells expressing Cx43-GFP. Annular gap junctions were observed to form both as a result of internalization of portions of gap junction plaques or entire gap junction plaques. In both cases the annular gap junction vesicle was released into one of two contacting cells ( Figure 2). We were unable to track or analyze the smaller gap junction vesicles since they quickly went out of focus. Of the annular gap junctions that were tracked, the average puncta size, measured from live cell imaging of Cx43-GFP expressing cells, was 1.6 ± 0.3 µm 2 , a size previously shown to be consistent with annular gap junctions and not secretory vesicles [19]. In Figure 2, a gap junction plaque is shown to first invaginate to produce a gap junction bud which in time detaches to form an annular gap junction vesicle. Over a 7 h viewing period 24.0 ± 3.4 annular gap junctions were observed to form.
As summarized in Figure 2H, over a 26 h monitoring period, 93 annular gap junctions, selected for tracking analysis, from four different movies, were observed to either (a) fragment to form smaller puncta, (b) join with cytoplasmic organelles, (c) disappear from view, or (d) move toward and appear to associate with the cell surface gap junction plaques. Note, while other puncta were observed to be released into the cytoplasm and remain relatively stationary, we did not quantitate their number. Since annular gap junctions, observed with FITC fluorescent and DIC imaging, were seen to join with other cytoplasmic organelles ( Figure 2B), we were prompted to determine the identity of the structures that associated with the annular gap junction vesicles.

Annular Gap Junction Fusion with Other Organelles
In cells expressing endogenous Cx43 as well as those transfected to express Cx43-GFP, annular gap junctions were observed, with immunocytochemical colocalization techniques, to associate with two different organelles, lysosomes ( Figure 3) as expected, and mitochondria ( Figures 4 and 5). The details of these interactions were analyzed. released into the cytoplasm and remain relatively stationary, we did not quantitate their number. Since annular gap junctions, observed with FITC fluorescent and DIC imaging, were seen to join with other cytoplasmic organelles ( Figure 2B), we were prompted to determine the identity of the structures that associated with the annular gap junction vesicles.

Annular Gap Junction Fusion with Other Organelles
In cells expressing endogenous Cx43 as well as those transfected to express Cx43-GFP, annular gap junctions were observed, with immunocytochemical colocalization techniques, to associate with two different organelles, lysosomes ( Figure 3) as expected, and mitochondria ( Figures 4 and 5). The details of these interactions were analyzed. The intimate contact between annular gap junction (AGJ) and lysosomes, which are in various stages of degradation is shown. In B (and enlarged in D) the outer membrane of the annular gap junction can be seen to be continuous with that of the lysosomal membrane (white arrows). Clathrin, identified by the bristle coat (arrows, C) or quantum dot label (black arrows, E), can be seen associated with annular gap junction vesicles that are fused with lysosomes. Typical clathrin-coated vesicles also can be seen decorated with clathrin Q dots (dashed white arrows in E). F (enlarged from G) and D (enlarged from B) have been magnified to allow the annular gap junction pentalaminar membranes to be clearly seen (black arrows) and the change in this membrane at the annular/lysosomal junction to be appreciated   The intimate contact between annular gap junction (AGJ) and lysosomes, which are in various stages of degradation is shown. In (B) (and enlarged in (D)) the outer membrane of the annular gap junction can be seen to be continuous with that of the lysosomal membrane (white arrows). Clathrin, identified by the bristle coat (arrows, (C)) or quantum dot label (black arrows, (E)), can be seen associated with annular gap junction vesicles that are fused with lysosomes. Typical clathrin-coated vesicles also can be seen decorated with clathrin Q dots (dashed white arrows in (E)). (F) (enlarged from (G)) and (D) (enlarged from (B)) have been magnified to allow the annular gap junction pentalaminar membranes to be clearly seen (black arrows) and the change in this membrane at the annular/lysosomal junction to be appreciated released into the cytoplasm and remain relatively stationary, we did not quantitate their number. Since annular gap junctions, observed with FITC fluorescent and DIC imaging, were seen to join with other cytoplasmic organelles ( Figure 2B), we were prompted to determine the identity of the structures that associated with the annular gap junction vesicles.

Annular Gap Junction Fusion with Other Organelles
In cells expressing endogenous Cx43 as well as those transfected to express Cx43-GFP, annular gap junctions were observed, with immunocytochemical colocalization techniques, to associate with two different organelles, lysosomes ( Figure 3) as expected, and mitochondria (Figures 4 and 5). The details of these interactions were analyzed. The intimate contact between annular gap junction (AGJ) and lysosomes, which are in various stages of degradation is shown. In B (and enlarged in D) the outer membrane of the annular gap junction can be seen to be continuous with that of the lysosomal membrane (white arrows). Clathrin, identified by the bristle coat (arrows, C) or quantum dot label (black arrows, E), can be seen associated with annular gap junction vesicles that are fused with lysosomes. Typical clathrin-coated vesicles also can be seen decorated with clathrin Q dots (dashed white arrows in E). F (enlarged from G) and D (enlarged from B) have been magnified to allow the annular gap junction pentalaminar membranes to be clearly seen (black arrows) and the change in this membrane at the annular/lysosomal junction to be appreciated

Analysis of Lysosomes/ Annular Gap Junction Associations
Lysosomes, detected with a lysosomal marker, Lamp 1, were shown to colocalize with annular gap junctions in cells expressing endogenous Cx43 ( Figure 3A). We counted the total number of connexin puncta and calculated the percentage that was observed to be in contact with lysosomes. Analysis of cytoplasmic puncta indicated that 8.3 ± 3.6% colocalized with lysosomes. In cells transfected to express Cx43-GFP there was a higher percentage of colocalization of annular gap junctions with the LAMP1 lysosome marker than that found in cells expressing endogenous Cx43. We believe the higher percentage colocalization with Cx43-GFP was due to an increased aggregation of tagged Cx43 in the cytoplasm of cells overexpressing the Cx43-GFP and thus the possibility of false positives.
A pitfall of interpreting colocalization data is that at the light microscopic level structures that are separated by as much as 200 nm can still appear to be colocalized. Further, the capacity to distinguish and analyze the morphology of the organelles is limited by the resolution of images collected with light microscopy. Transmission electron microscopy was used to better resolve the morphology and to positively identify organelles and their associations.
The annular gap junction vesicle pentalaminar or dense double membrane was used to identify gap junction structures with TEM ( Figures 1D-F and 3B-G). Taking advantage of the morphological characteristic of the annular gap junction membrane and the well-known distinguishing characteristics of other cytoplasmic organelles, we thus analyzed the association of organelles with gap junction structures.
Annular gap junctional vesicles were observed to interact with lysosomes ( Figure 3). Lysosomes were identified by their limiting membrane which is single, unlike that of annular gap junction vesicle and mitochondria, and the density of the material found within their lumen. Lysosome morphology however was variable and the density of the material within their lumen was dependent on the stages of digestion ( Figure 3B-G). In annular gap junctional profiles that appeared to be undergoing degradation, the inner and outer annular gap junction membranes were separated from one another and the outer membrane was continuous with the lysosomal membrane ( Figure 3B,D), as if the two distinct organelles were becoming one. Clathrin was found associated with the annular gap junction,

Analysis of Lysosomes/Annular Gap Junction Associations
Lysosomes, detected with a lysosomal marker, Lamp 1, were shown to colocalize with annular gap junctions in cells expressing endogenous Cx43 ( Figure 3A). We counted the total number of connexin puncta and calculated the percentage that was observed to be in contact with lysosomes. Analysis of cytoplasmic puncta indicated that 8.3 ± 3.6% colocalized with lysosomes. In cells transfected to express Cx43-GFP there was a higher percentage of colocalization of annular gap junctions with the LAMP1 lysosome marker than that found in cells expressing endogenous Cx43. We believe the higher percentage colocalization with Cx43-GFP was due to an increased aggregation of tagged Cx43 in the cytoplasm of cells overexpressing the Cx43-GFP and thus the possibility of false positives.
A pitfall of interpreting colocalization data is that at the light microscopic level structures that are separated by as much as 200 nm can still appear to be colocalized. Further, the capacity to distinguish and analyze the morphology of the organelles is limited by the resolution of images collected with light microscopy. Transmission electron microscopy was used to better resolve the morphology and to positively identify organelles and their associations.
The annular gap junction vesicle pentalaminar or dense double membrane was used to identify gap junction structures with TEM ( Figure 1D-F and Figure 3B-G). Taking advantage of the morphological characteristic of the annular gap junction membrane and the well-known distinguishing characteristics of other cytoplasmic organelles, we thus analyzed the association of organelles with gap junction structures.
Annular gap junctional vesicles were observed to interact with lysosomes ( Figure 3). Lysosomes were identified by their limiting membrane which is single, unlike that of annular gap junction vesicle and mitochondria, and the density of the material found within their lumen. Lysosome morphology however was variable and the density of the material within their lumen was dependent on the stages of digestion ( Figure 3B-G). In annular gap junctional profiles that appeared to be undergoing degradation, the inner and outer annular gap junction membranes were separated from one another and the outer membrane was continuous with the lysosomal membrane ( Figure 3B,D), as if the two distinct organelles were becoming one. Clathrin was found associated with the annular gap junction, even at the point that they were fused with lysosomes ( Figure 3C,E). With ultrastructural analysis the number of lysosomal/annular vesicle associations could be quantitated in detail. Based on the analysis of 549 annular gap junction vesicles, we demonstrated that 2.6 ± 0.6% were fused with lysosomes.

Mitochondria
In addition to colocalization with lysosomes, annular gap junctions were revealed with immunocytochemistry, super-resolution microscopy, and transmission electron microscopy (TEM) to colocalize with mitochondria ( Figure 4). With immunocytochemistry the size of the puncta was used to identify a structure as an annular gap junction ( Figure 4A). In Figure 4, colocalization of mitochondria detected with either antibody to Mitotracker (red) or TOM (red) and annular gap junctions detected with Cx43 antigen (green) was evident. As seen with super-resolution microscopy some mitochondria colocalize with more than one annular gap junction vesicle.
To visualize the fine structure morphology at the sites of physical association between the mitochondria and annular gap junctions, TEM imaging was used ( Figures 4C and 5). Mitochondria were observed to associate and in some cases to follow the contour of annular gap junction membranes ( Figures 4C and 5). Actual direct fusion of the outer membrane of the annular gap junction vesicles and mitochondria were sometimes evident ( Figure 5). We selected a distance of less than 25 nm as indicative of contact area. This distance was selected based on the reported contact distance of 10-30 nm between mitochondria and surface gap junction plaques [43] as well as lysosomes [44,45]. The distances, all less than 25 nm, were averaged and the data is expressed as standard error from the mean. The average contact distance between mitochondria and annular gap junctions was 18.4 ± 0.7 nm with a range from 0-25 nm. The distances were calculated from four different TEM preparations with N = 200.
Ultrastructural analysis of 549 images of annular gap junctions revealed that 5.2 ± 1.1% associated with mitochondria. The finding that annular gap junction vesicles appeared to be in direct contact with mitochondria, was significantly greater than expected by random chance. The percentage of annular gap junction vesicles in contact with mitochondria (5.2 ± 1.1%) exceeded the percentage of annular gap junction vesicles that were found to associate with lysosomes (2.6 ± 0.6%).
To address the question of random vs. specific interaction of annular gap junctions and mitochondria, a Wilcoxon rank-sum nonparametric test was used to compare the mean number of annular gap junction/mitochondria contacts vs. annular gap junction vesicle/vacuoles contacts. Over a randomly selected 20 images, there were 188 total mitochondria and 240 total vacuoles. The number of mitochondria per image ranged from 0 to 39 with a mean of 9.4 ± 0.75, while the number of vacuoles ranged from 0 to 35 with a mean of 12 ± 0.65. In 35% of the images, there was at least one annular gap junction in contact with a mitochondrion while only one image indicated an annular gap junction contacting a vacuole. The mean number of annular gap junctions in contact with mitochondria was 0.346 ± 0.078 vs. 0.009 ± 0.007 in contact with a vacuole. A Wilcoxon rank-sum nonparametric test comparing the mean number of touches per image resulted in a statistically significant higher rate of annular gap junctions touching a mitochondria than a vacuole (p = 0.008), consistent with specific rather than random events.
To further address the concerns of random vs. nonrandom chance contact based on annular gap junction sizes, we measured annular gap junction profile sizes and compared these measurements to the average distance between mitochondrial membranes. If annular gap junction contact with mitochondria was random, then we would expect larger annular gap junctions to more frequently contact mitochondria than smaller ones. We found the average gap junction profile measured with TEM was 421 ± 16.9 nm (n = 199 annular gap junction). We arbitrarily selected 365nm, the median annular gap junction size, for classifying annular gap junctions as either large or small. Based on chi square analysis we saw no size-based difference (p = 0.83) in the frequency of finding a large (size 596.8 ± 21.7 nm) annular gap junction contacting a mitochondria (19, n = 100) than a smaller (size 244.2 ± 71.6 nm) annular gap junction contacting a mitochondria (19, n = 99). The average contact distance between mitochondria and the larger (18.7 ± 1.0 nm) or smaller (17.9 ± 0.9 nm) gap junctions was not significantly different, as calculated with a Student's t-test (p = 0.57).
In images in which annular gap junctions were in the same field, the average minimal distance between the annular gap junction and the nucleus was 330.9 ± 96.7 nm. That the annular gap junction vesicles were not in contact with the relatively large nucleus would be consistent with the interaction of annular gap junctions with the mitochondria not being a random encounter based on the size of the annular but that a specific interactions between mitochondria and annular exist.

Discussion
In this study, we documented the presence of cytoplasmic Cx43-containing structures and we analyzed the details and frequency of interactions of mitochondria and lysosomes with annular gap junction vesicles. We found that the interactions between annular gap junctions and mitochondria were seen more frequently than between annular gap junctions and lysosomes. The behavior of the puncta believed to be annular gap junctions were analyzed with live cell imaging and it was determined that these structures could either (1) undergo fission [23], (2) fuse with other organelles, (3) disappear from view, or (4) appear to recycle back to the surface. In this study, to address the concern that in our live cell imaging studies GFP tag may either interfere with or alter organelle interactions with Cx43-containing structure, or that the Cx43 puncta were not positively identified as annular gap junction vesicles, we took advantage of TEM techniques to positively identify and analyze organelle association in cells expressing endogenous Cx43.
It was determined, based on ultrastructural analysis of the structures in the cytoplasm, that some of the puncta identified as annular gap junction at the light microscopic level of resolution could be cytoplasmic aggregates. It should be noted however that such structures were relatively rare, and thus our findings would be consistent with most of the puncta seen with live cell imaging being annular gap junctions.
It is well accepted that connexin in annular gap junction vesicles are degraded by lysosomal, proteasomal, or autophagosomal pathways [14,24,[42][43][44][45]. Lysosomal degradation is based on TEM observations of colocalization of lysosomes and annular gap junction [8,14], the increase in annular gap junction vesicles when lysosomal activity is inhibited [44], and the demonstration of acid phosphatase activity in these annular gap junctions [8,14]. In this current study we did not analyze proteasomal or autophagosomal pathways but instead have characterized the most common degradation pathway, lysosomal degradation. We found with TEM, that only 2.6% of the annular gap junction vesicles analyzed were associated with lysosomes at a given point in time. Although this figure may suggest a limited contribution to the gap junction turnover or behavior, any alteration in the rate of Cx43 degradation might serve as a post-translational means of altering intercellular communication. While the association of lysosomes with annular gap junction appeared as expected to result in the fusion of the two organelles, the contact between mitochondria and annular gap junction vesicles did not result fusion to form one structure.
Although the close association of surface gap junction plaques and mitochondrial has been reported [43,46,47] the association of annular gap junction with mitochondria, to our knowledge, has not been previously characterized. Forbes and Sperelakis showed an annular structure that could be an annular gap junction that is associated with a mitochondrion [43]. However, this early study was before live cell imaging of tagged Cx43 was readily available and the authors speculated that the annular profile was an invaginated surface gap junction cut at an angle that made it appear to be annular. Although the association of mitochondria and annular gap junctions was not mentioned by Emdad and colleagues, an image demonstrating contact between these two organelles was seen in a TEM image in this paper [48]. The authors suggested the annular profiles could represent either sectioning through convoluted gap junction plaques or internalized annular gap junctions. In the current study we characterize the contact between annular gap junctions and mitochondria with TEM, immunocytochemistry, and super-resolution microscopy.
The contact between mitochondria and annular gap junctions viewed with TEM was a distance as close as 25 nm or less at the site of contact and in some cases the two membranes of the two organelles were actually in physical contact with one another. This contact distance is comparable to the reported distance of 10-30 nm measured between the mitochondria and gap junction plaques [43], lysosomes [44,45], or the endoplasmic reticulum (ER) [45]. The existence of ER-mitochondria contact sites has been well established by electron microscopy and time-lapse fluorescence microscopy and it has been suggested that in yeast, a network of contact sites serve to integrate the ER, vacuoles, and mitochondria [45]. Here we suggest that in addition to the interaction between ER and vacuoles, mitochondria also interact with annular gap junction vesicles. The benefit of this interaction can only be speculated at this point but it is tempting to suggest one possibility may be that Cx43 from annular gap junctions may be delivered to mitochondria when the two organelles come into physical contact. Future studies are needed to confirm this speculation. In addition to Cx43 from the annular gap junction being delivered to the mitochondrial membrane, it is also possible that connexins may arrive at the mitochondria in secretory vesicles from Golgi or ER interactions. We have not ruled out these possibilities.
It has been well documented however that there is Cx43 on mitochondrial membranes [49][50][51][52]. Proof of mitochondrial Cx43 have been provided from investigators who used a wide variety of different techniques, including immunocytochemical colocalization of Cx43 with mitochondrial proteins at the light microscopic and immunogold electron microscopic levels of resolution [49], flow cytometry studies, and quantitative Western blot analysis [49][50][51][52]. Mitochondrial Cx43 has been demonstrated to protect the heart from ischemic injury, facilitating mitochondrial movement to the cell periphery, as well as maintaining mitochondrial network integrity [49,50,[53][54][55][56][57][58]. Further, mitochondrial metabolic activity and morphology alterations have been demonstrated in adipocytes from Cx43 knockout mice and knockdown cells in culture [54], consistent with there being a Cx43 mitochondrial functional dependency [49,56,57]. Additionally, since mitochondria are known to sequester calcium ions and to provide cell energy, it is possible that annular gap junction contact with mitochondria may function in regulating annular gap junction channels and/or ATP exchange.
Intervention studies that measure changes in mitochondria Cx43 relative to treatments that alter annular gap junction formation or degradation and thus increasing or decreasing annular gap junction number available to interact with the mitochondria are needed. Further, examining cell types that have different levels of gap junction plaque internalization would be useful. As to why some cell types internalize gap junctions more often than others is not clear, however there are a number of interventions that will alter this internalization process including treatments that modulate protein kinase expression [59,60]. For example, elevating Src and protein kinase C increases frequency of internalization [60], but elevating protein kinase A decreases internalization [61,62]. It is thought that this change in the frequency of internalization and thus annular gap junction formation is regulated by kinase mediated phosphorylation of the Cx43 proteins [61,62]. Treatments with kinase activators, inhibitors or the study of cells expressing the truncated or mutated Cx43 as well as those with autophagosomal markers or proteasomal markers are needed to increase our understanding of Cx43 phosphorylation and annular gap junction interactions with cytoplasmic organelles. Further, we have demonstrated that internalization involves clathrin and dynamin mediated mechanisms that if perturbed alters annular gap junction formation [23]. Zonula occludens-1 protein (ZO-1) has been demonstrated to bind to the C-terminal tail of Cx43 where it contributes to a number of Cx43 functions including, gap junction channel formation, docking and lateral movement to determine gap junction plaque size [9, [63][64][65][66]. Cx43/ZO-1 binding is regulated via phosphorylation/de-phosphorylation of S373 by AKT kinase [67]. Src-mediated dissociation of ZO-1 initiates endocytic internalization of gap junctions and thus annular gap junction formation [64]. Gap junction plaque size has been demonstrated to be modulated by ZO-1 binding and it has been reported that tagging GFP to the C-terminal of Cx43 inhibits ZO-1 binding [64,68], increases gap junction plaque size and endocytosis resulting in increased size and number of annular gap junctions [64]. In contrast in the human heart, diminished ZO-1 levels coincide with reduced Cx43 staining [69]. The relationship between ZO-1 and gap junction size may vary depending on tissue types and pathological conditions. The SW-13 cells used in this study form large gap junctions [8]. While ZO-1 levels or the effect of expressing Cx43-GFP on annular organelle contacts or gap junction plaque size were not studied, at least for these cells that express only endogenous Cx43, the annular size does not appear to influence the number of mitochondria/annular gap junction contacts. In an early study, in which the occurrence of annular gap junction in various cell types was reviewed, it is apparent that some cell types have more annular gap junctions than other cell types [70]. While the SW-13 appear to form large gap junctions that are frequently internalized relative to many other cell types [8,70], there are no or few reports comparing annular gap junction formation in different cell types. The benefits of some cells having larger gap junctions are unknown. It is however presumed that these cells may be more dependent on cell-cell communication to maintain function. For example, gap junction plaques have been shown in vivo and in vitro to be larger in the ventricle than in atrial myocytes [69,71]. The function of large ventricular gap junctions has been speculated to play a critical role in intercellular current transfer, needed for normal ventricular function. Further, annular gap junction profiles increased when hypertrophy of the left ventricle was induced by abdominal aorta banding [48]. How the number of annular gap junctions in cells that have fewer or more annular gap junctions or that make larger or smaller surface gap junctions affects mitochondria function is unclear and can only be speculated. The factors regulating cell-specific frequency of internalization, annular gap junction vesicle size, and their subsequent interactions with mitochondria and lysosomes need to be investigated in a variety of cell types if we are to fully understand gap junction protein trafficking.
The method of Cx43 delivery to the mitochondria is not fully understood however it appears to involve heat shock protein 90 (Hsp90) and TOM 20 [39]. Further, it is known that mitochondrial Cx43 is phosphorylated [58], however it is not known which Cx43 amino acid residues are phosphorylated. The mechanism that regulates trafficking of Cx43 to the mitochondria and its protective role in pathophysiological conditions remain unknown. Is the annular gap junction vesicle possibly a cytoplasmic source of phosphorylated Cx43 that could potentially serve as a source for Cx43 distribution to other compartments? Although it is well accepted that connexins in annular gap junctions are degraded, other possible fates have received little attention despite the reports of Cx43 recycling from annular gap junctions to participate in rapid gap junction plaque [31,32,64,72].
The method by which Cx43 traffics to the mitochondria has not been elucidated here. However, the findings of Cx43 targeting to the mitochondria and Cx43-dependent changes in mitochondrial function and movement within the cytoplasm [49,50,[53][54][55][56][57][58] points to the possibility that in addition to traditional Cx43 trafficking and function at the cell surface that Cx43 may traffic and function in noncanonical manners. Here we demonstrate the association of annular gap junctions with mitochondria and suggest that Cx43 in annular gap junctions may have a fate that is unrelated to degradation. Future studies are needed to elucidate the 'tethering' molecules that hold annular gap junctions to mitochondria and to clarify the need for the interaction annular gap junctions with mitochondria.

Gap Junction Antibodies and Probes
Affinity purified polyclonal rabbit antibodies (IgG) were prepared against synthetic peptides corresponding to the carboxyl terminus of the Cx43 molecule (residues 370 to 381) [73] (Zymed Laboratory, San Francisco, CA, USA). Preparation and characterization of these antibodies have been previously described [73].

Immunocytochemistry
Cells were prepared and stained with immunocytochemical techniques, as previously described [23] for connexin 43 (1:100; Proteintech, Rosemont, IL, USA), clathrin (Abcam, Cambridge, MA, USA), lysosomes with Lamp1 staining (Abcam), and mitochondrial proteins with Mitotracker (1:100) or Tom20 (Santa Cruz, Dallas, TX, USA) (1:1000). Prior to immunocytochemical procedures, the cells were grown on coverslips, rinsed with phosphate buffered saline solution (PBS), fixed at room temperature, in 3% formaldehyde for 20 min, permeabilized in cold acetone for 7 min, and then incubated at 37 • C for 1 h or at 4 • C overnight in the primary antibody. In some experiments colocalization studies where performed. In these procedures, labeling of connexin was followed by a 4 • C overnight incubation in clathrin antibody. In experiments to colocalize mitochondria, the cellular localization of Tom-20, or mitotracker was visualized with a rhodamine conjugated goat anti-mouse secondary antibody (1:1000; Pierce, Rockford, IL, USA). The coverslips were stained with Hoescht, washed in PBS, and placed onto glass slides with a drop of Fluoromount-G antiquench reagent (Southern Biotechnical Lab, Birmingham, AL, USA). Immunolabeled cells were imaged at 63X with an Olympus Provis microscope (Olympus, Center Valley, PA, USA) and with the MetaMorph Imaging System (Molecular Devices, Downington, PA, USA).
For immunocytochemical analysis, the number of gap junction plaques and annular vesicles per cell nuclei were calculated. Fluorescent spherical puncta that were greater-or-equal to 0.5 µm in diameter were classified as annular gap junction vesicles while plaques were identified by their typical elongated profile or by the presence of aggregated puncta (smaller plaques), that were obviously at the cell surface between two cell pairs. The data was expressed as the average number of annular gap junction vesicles per cell.

Percent Colocalization
The percent colocalization was calculated from 50 cells (10 cells were selected from each of five files) in images collected by confocal step through selecting elements at the mid-plane of the Z stack. All images were saved as tiffs prior to analysis. The MetaMorph Software Program was used to view and trace the cell borders and to determine the percent colocalization of cells positive for both signals. Specifically the merged image was separated into red and green channels and the trace areas were transferred to the separated images. After thresholding the images, the number of Cx43 containing objects was determined with the automatic counting tool in MetaMorph. The counts were displayed on the "record count screen", which was then analyzed and the number of surface gap junctions (as well as the number of clusters or aggregates or any object that did not look like an annular gap junction) were counted manually and subtracted from the total number of objects. The number of overlapping areas (yellow objects) were then counted manually by analyzing the merged image. The number of annular gap junctions that colocalized with lysosomes or mitochondria were determined by counting the number of annular gap junctions that had any yellow associated with them. The data was expressed as the average percent colocalization of annular gap junction vesicles with lysosomes or mitochondria.

Transfection with cDNA
To visualize gap junction trafficking in living cells, adrenal cells were transfected with cDNA encoding the fluorescent Cx43-GFP (provided by Dr. M. Falk, Lehigh University, Bethlehem, PA, USA). The Cx43-GFP vector was constructed by linking the GFP fluorescent reporter protein to the C-terminus of the rat Cx43 cDNA, and it has been demonstrated to assemble into gap junction plaques similar to wild type Cx43 [23]. Lipofectamine 2000 Transfection Reagent (Life Technologies, Grand Island, NY, USA) was used to establish cell populations that transiently expressed fluorescently tagged Cx43, GFP, or empty vector.
The day before transfection, cells were seeded onto coverslips or in Mattek cell culture dishes (MatTek Corporation, Ashland, MA, USA). Cell populations, at 70-80% confluence, were transfected in Opti-mem medium which contained Lipofectamine 2000 Transfection Reagent (Invitrogen) and 4 µg of plasmid DNA (Cx43-GFP, or empty vector) for 4 h at 37 • C in an atmosphere of 5% CO 2 . The resulting complexes were removed by gentle aspiration and washed with PBS. Fresh L-15 complete cell growth medium was added to the dishes, and the cells were incubated at 37 • C in 5% CO 2 for 24-48 h before imaging.

Imaging of Cx43-GFP in Living Cells
MatTek glass bottom dishes with cells expressing Cx43-GFP or empty vector were placed into a temperature controlled chamber and maintained at 37 • C in 5% CO 2 on a live cell Nikon A1 series automatic confocal laser point scanning system (Nikon Instruments Inc., Melville, NY, USA). Image acquisition on the Olympus was performed with a MetaMorph Imaging System (Molecular Devices) and Nikon NIS-Elements on the A1 Nikon microscope.
Images obtained with a 63× oil objective with a numerical aperture of 1.4, were collected at 1 min intervals over a 26 h viewing period. DIC and fluorescent images were obtained using the standard fluorescence detector at wavelengths 482 and 595 for all experiments. Focus was maintained with the Perfect Focus System (PFS) function and images were acquired with Nikon's NIS-Elements software. Qualitative and quantitative analyses were performed on the data sets with the rendering tool in NIS-Elements. Time-lapse images were used to analyze gap junction plaque internalization and for analysis. Time-lapse images were converted into movies and evaluated with the MetaMorph program (Molecular Devices).
Annular gap junction vesicle pattern of displacement within the cell and corresponding changes in size (area expressed as µm 2 ) were quantitated with the tracking function in the Imaris analysis software (Bitplane Scientific, South Windsor, CT, USA). Selected annular vesicles were segmented based upon labeling intensity and then followed over time. Statistical significance of differences was determined with the Student's t-test.

Super-Resolution Live Cell Microscopy
Super-resolution microscopy was used to analyze annular gap junction and mitochondrial interaction with the stimulated emission depletion (STED) microscopy technique. STED allows for enhanced spatial resolution of the mitochondria which improves our accuracy when analyzing annular gap junction associations with mitochondria across the x, y, and z planes. Gated STED images were obtained using a commercial Leica SP8 STED 3X system (Leica Microsystems, Mannheim, Germany) housing a 100× oil objective with a numerical aperture of 1.4. Connexin 43 and Tom-20 antibodies were used to label annular gap junctions and mitochondria, respectively, for STED imaging.

Transmission Electron Microscopy
Cell monolayers were briefly rinsed in PBS then fixed with 2.5% glutaraldehyde in PBS, pH 7.4, for 1 h at room temperature. All samples were then washed 3 times in PBS buffer and post-fixed for 1 h at 4 • C in 1% osmium tetroxide with 1% potassium ferricyanide. The samples were washed again and the cells were serially dehydrated in an ethanol (30%, 50%, 70%, and 90%) for 10 min followed by 15 min in 100% ethanol three times. The cells were then incubated in Epon 3 times for 1 h and finally embedded in resin to be sectioned. Ultrathin sections were cut, mounted on grids, and imaged on a JEOL 1011CX electron microscope (JEOL, Tokyo, Japan).
In Quantum dots protocols and in some protocols to view annular gap junction/organelle contacts, the cells were fixed (2% paraformaldehyde and 0.1% glutaraldehyde in PBS). Quantum dots (QDs) were used to specifically label connexin and clathrin. Adrenal cells were incubated in Cx43 or clathrin primary antibodies, followed by incubation in biotin IgG conjugated antibody, and then in a QD conjugated secondary antibody (Strepavidin conjugated with QD 655).
Two procedures were used to test the specificity of the quantum dot immuno-electron staining. First, the cells were incubated in the quantum dot-linked anti-rabbit immunoglobin without a preincubation of the thin sections in the first antibody. Second, the binding of quantum dot complexes to embedding resin was evaluated in areas free of cells.

Statistical Analysis
Data were analyzed with an unpaired two-tailed Student's t-test, chi-squared test, or one-way analysis of variance (ANOVA). Wilcoxon rank-sum nonparametric test was used to compare the percentage of mitochondria/annular gap junction vesicles vs annular gap junction/vacuole contacts versus the percent expected by random chance. Data are presented as means ± SEM. In this analysis, out of 20 randomly selected images, there were 188 total mitochondria and 240 total vacuoles. The number of mitochondria per image ranged from 0 to 39 with mean = 9.4, while the number of vacuoles ranged from 0 to 35, mean = 12. The number of annular gap junctions was 0, 1, or 2. There were 10 images with at least one mitochondria and one annular gap junction and 11 images with at least one vacuole and annular gap junctions. These were used to compare annular gap junction contacts with mitochondria vs. vacuoles.


extfieldsofstudy: Biology Medicine

text:
Highly expressed placental miRNAs control key biological processes in human cancer cell lines

Despite being a healthy tissue, the constituent cells of the placenta, share similar characteristics with tumor cells, such as increased cell growth, migration, and invasion. However, while these processes are stochastic and uncontrolled in cancer cells, in placenta they are precisely controlled. Since miRNAs have been reported to regulate genes that control the molecular mechanisms necessary for the development of both human placenta and cancer, we addressed for miRNAs highly expressed in the placenta that could be involved in tumorigenesis. Here, we assessed the miRNA profile in placenta samples using microarray analysis. The results showed that miR-451 and miR-720, highly expressed placental miRNAs, presented very low or undetectable expression in cancer cell lines compared to the normal placenta and healthy tissues. Additionally, transfection of miR-451 or miR-720 mimics in choriocarcinoma cell line (JEG3) and colorectal adenocarcinoma cell line (HT-29) resulted in impaired cell proliferation, decreased cell migration and invasion and reduced ability of colony formation. These findings provide evidence that placenta may work as an alternative model to identify novel miRNAs involved in pathways controlling tumorigenesis.

INTRODUCTION
Human placenta is the most specialized transient organ of the pregnancy, which together with the fetal membranes and the amniotic fluid supports the normal growth and development of the embryo [1]. Both the structure and function of the placenta are dictated by the need to maintain fetal growth, keeping the balance between the fetal and maternal systems. Therefore, during pregnancy, the human placenta undergoes a dramatic structural reorganization to functionally synchronize the development of the embryo and the maternal compartment [2]. The development of the placenta is a critical step for the normal growth of the embryo. Trophoblastic cells are responsible for the ability of invasion and proliferation of the placenta [3], properties similar to those associated with the development of cancer. Thereby, despite being a healthy tissue, the constituent cells of the placenta,

Research Paper
especially trophoblasts, share similar characteristics with tumor cells, such as increased cell growth, migration, and invasion [3,4]. However, while these processes are stochastic and uncontrolled in cancer, the cells of the placenta present regulatory mechanisms that precisely control cell growth, migration, and invasion of the uterine tissue [2,5]. Loss of control of these biological processes may result in the development of choriocarcinoma, a highly malignant disease [6].
MicroRNAs (miRNAs) are small non-coding RNA molecules (19-22 nucleotides) that act in the posttranscriptional regulation of gene expression. In general, this regulation occurs through the binding of a specific miRNA region known as seed (2-8nt in the 5′ region of miRNA) to the 3′ UTR (untranslated region) of the target mRNAs [7,8]. Several reports have shown that miRNAs play a crucial role in the development of many diseases, including placenta-associated diseases and cancer. Some studies have shown that the development of preeclampsia is associated with altered expression of placental miRNAs [9,10]. Furthermore, changes in miRNA expression profiles have also been described for several cancers, including breast cancer [11], leukemia [12], prostate cancer [13], among others. Therefore, miRNAs may regulate genes controlling the molecular mechanisms necessary for the development of both human placenta and cancer, such as proliferation, migration, invasion, and apoptosis.
Since the development of placenta shares key molecular mechanisms with the development of cancer, placenta becomes an ideal model to identify novel miRNAs that can be involved in tumorigenesis. For this purpose, we assessed the miRNA expression profile of normal human placenta and evaluated the biological function of highly expressed placental miRNAs in cancer cell lines. From the top five highly expressed placental miRNAs, we demonstrated that miR-451 and miR-720 were highly expressed in normal human placenta and several other normal tissues, but were downregulated in cancer cell lines. Restoration of expression indicates a possible role for miR-451 and miR-720 in controlling proliferation, migration, and invasion at least in choriocarcinoma and colorectal cancer cells.

RESULTS
Highly expressed miRNAs in the normal human placenta miRNA expression analysis was performed in the human placentas ranging from 37 to 40 weeks of gestation (two samples representing each gestational age). RNA quality analysis revealed an average RIN of 6.06 (ranging from 5.1 to 6.7).
Therefore, highly expressed miRNAs in placenta was considered as the ones that presented expression (signal intensity) above the upper limit (outliers: points 1.5 IQR above the 3rd quartile) according the distribution of the microarray expression data in the boxplot. This approach allowed the identification of 235 miRNAs highly expressed in normal human placenta, from which we selected the 30 most highly expressed miRNAs ( Figure 1 and Supplementary Table 1). miR-451 and miR-720 are highly expressed in normal human placenta and normal tissues but downregulated in cancer cell lines Among the top-five most highly expressed miRNAs in normal human placenta, we evaluate the expression of miR-451, miR-517a, miR-517b and miR-720 in normal human placenta samples, several normal human tissues and different cancer cell lines using RT-qPCR. miR-923 was excluded from the analysis, because it was reported as a fragment of ribosomal RNA.
Our results confirmed miR-451 and miR-720 elevated expression levels in all normal human placenta samples, corroborating the microarray data. Additionally, we demonstrated that the expression of both miRNAs was not restricted to the normal placenta. miR-451 expression was also observed in several human normal tissues, presenting equal or higher expression levels in comparison to the normal placenta ( Figure 2A). Similarly, miR-720 expression was also observed in several human normal tissues. However, its expression was lower as compared to the placenta samples ( Figure 2B). Interestingly, for both miRNAs, we noticed that their expression in cancer cell lines was very low or undetectable ( Table 2).
For miR-517a and miR-517b, despite the fact that we observed their higher expression in placenta samples, both presented very low or undetectable expression in all the human normal tissues evaluated (data not shown). For this reason, these miRNAs were not considered for the further analysis. miR-451 and miR-720 control key biological processes in cancer cell lines miR-451 and miR-720 were expressed in normal tissues, including placenta, but downregulated in cancer cell lines as compared to normal samples, suggesting that both miRNAs could be important to the maintenance of the normal phenotype in placenta cells and normal tissues. Therefore, we assessed whether these miRNAs could be involved in the regulation of cellular biological processes critical for tumor development. Using miRNA mimics, we overexpressed miR-451 or miR-720 ( Figure 3A and 3B) in a cancer cell line derived from choriocarcinoma (JEG3) and a colon adenocarcinoma cell line (HT-29), both expressing low levels of those miRNAs (data not shown).
Our data demonstrated that miR-451 or miR-720 ectopic expression impaired cell proliferation in both www.oncotarget.com JEG3 and HT-29 cancer cell lines ( Figure 4). Additionally, we observed that overexpression of miR-451 or miR-720 dramatically decreased cell migration in both cell lines ( Figure 5A). JEG3 cells also had their invasion ability impaired upon overexpressed miR-451 or miR-720 ( Figure 5B). On the other hand, HT-29 cells did not show invasiveness ability, even when we used more cells/well or maintained the experimental conditions for longer periods (data not shown). Furthermore, colony formation assay demonstrated that miR-451 or miR-720 overexpression significantly reduced the ability of both JEG3 and HT-29 cells to establish colonies after twelve days of culturing ( Figure 5C).

DISCUSSION
Some miRNAs have been described to be exclusively expressed in human placenta [14,15]. Most of them are identified in maternal plasma during pregnancy [16]. Such observations suggest that miRNAs may play an important role in maternal-fetal communication, possibly promoting maternal adaptation to pregnancy [15,17]. Moreover, miRNA differential expression in maternal plasma has been used as a marker to predict complications during pregnancy, such as preeclampsia [18,19].
The decreased expression of placental miRNAs has also been described, contributing to the regulation of tumor invasion [20], cell proliferation, migration and differentiation [21]. Recently, our group demonstrated that the restoration of the expression of placenta-enriched long intergenic non-coding RNAs (lincRNAs) was associated with a decrease in cell migration and invasion of the JEG-3 cell line [22].
In this report, we demonstrated that miR-451 and miR-720 highly expressed placental miRNAs, presented very low or undetectable expression in cancer cell lines when compared to the normal placenta and other normal tissues. Additionally, ectopic expression of miR-451 or miR-720 in choriocarcinoma cell line (JEG3) or colon adenocarcinoma cell line (HT-29) resulted in impaired cell proliferation, decreased cell migration and reduced ability of colony formation in both cells lines. Also, it was associated with a reduction in the invasion ability of JEG3 cancer cells.
Scientific literature regarding miR-451 and miR-720 expression in human placenta is scarce. However, miR-451 and miR-720 elevated expression in human placenta samples has already been demonstrated in previous studies [15]. Additionally, the differential expression of miR-451 was related with placenta undergoing hypoxic conditions [23]. MiR-451 was suggested as a tumor suppressor as it has been associated with the regulation of several biological processes, including cell proliferation, migration, invasion and treatment response in glioblastoma [24,25], colorectal carcinoma [26,27] and lung cancer cell lines [28,29]. MiR-451 low expression was also associated with cell survival and resistance to hormone therapy in patients with breast carcinoma [30,31] and with the progression and worse prognosis in osteosarcoma [32,33].
Although few reports are available, data have been shown to be controversial regarding miR-720 expression in several tumor types. In accordance with our findings, some have reported miR-720 among miRNAs with lower expression levels in renal carcinoma [34], gastric lymphomas [35], pancreatic carcinoma [36] and esophageal carcinoma [37]. Also, miR-720 low expression was observed in lung carcinoma patients with poorer survival [38] while restored expression was associated with inhibition of tumor invasion and migration of breast cancer [39]. Nonetheless, miR-720 has also been demonstrated with increased expression in myelodysplastic syndrome [40], multiple myeloma [41], colorectal carcinoma [42] and melanoma [43]. Our data, together with these observations suggest that the role of miR-720, as an oncogene or tumor suppressor, depends on the cellular context, which has also been demonstrated for other miRNAs [44].  In summary, our results highlight the human placenta as an applicable model for the study of tumorigenesis. Furthermore, we demonstrated that the evaluation of highly expressed miRNAs in human placenta is a powerful strategy for the identification of miRNAs involved in the regulation of important biological processes in cancer cells. Together, our data indicate a possible role for miR-451 and miR-720 in controlling proliferation, migration, and invasion at least in choriocarcinoma and colorectal cancer cells. Future studies are necessary to gain further mechanistic insights into miR-451 and miR-720 role in tumorigenesis and assess if these findings can be translated to clinical application.

Human normal placenta samples
A total of 21 normal human placenta samples (35 to 40 weeks) were collected at birth by cesarean or vaginal delivery from healthy women (age range from 21 to 33, mean: 27,7; years). We excluded placentas from women presenting hypertension, preeclampsia, placenta previa, preterm labor, diabetes, autoimmune disease or infections (HPV, HIV, and others). The samples were collected at the obstetric center of the Clinics Hospital of the Ribeirão Preto Medical School -University of São Paulo (HC-FMRP/USP), and at the Woman Health Reference Center of Ribeirão Preto (CRSM-Mater).
The study was conducted following the national and institutional ethical policies and was previously approved by the Institutional Ethical Committee from both institutions (protocol # 9411/2010 for HC-FMRPUSP; and 009/2010 for CRSM-Mater). All the pregnant women included in the study agreed to participate by signing a specific consent form.

Human cancer cell lines and normal tissues
We evaluated a total of 16 human cancer cell lines comprising nine different human tissues:   [45]. Genotyping confirmed the identity of all cell lines.
We also used a commercial panel of total RNAs extracted from 20 different normal human tissues, FirstChoice Human Total RNA Panel Survey (Ambion, Austin, TX, USA).

RNA extraction and quality assessment
The total RNA from normal placenta samples and human cancer cell lines was isolated using TRIZOL (Invitrogen, Grand Island, NY, USA), according to the manufacturer's instructions [15]. RNA was quantified using Nanodrop 1000 Spectrophotometer (ThermoFisher Scientific, Waltham, MA USA). RNA quality was assessed using the Eukaryote RNA Total Pico kit (Agilent Technologies, Santa Clara, CA, USA) in Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA), according to manufacturer's instructions.

miRNA expression in normal placenta samples
miRNA expression profile of normal placenta was performed with Human miRNA Microarray Version 3 platform (cat# G4470C, Agilent Technologies, Santa Clara, CA, USA) and miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, CA, USA), according to manufacturer's instructions. The platform contains specific probes for the expression analysis of 866 human miRNAs described in public databases (miRBase release 12.0) [46]. Microarray slides were scanned using the High-Resolution Microarray Scanner (Agilent Technologies, Santa Clara, CA, USA). Captured images were processed, and raw data were extracted by the Agilent Feature Extraction Software v.8.5 (Agilent Technologies, Santa Clara, CA, USA).

Microarray analysis
Raw data files from miRNA expression for each of the human normal placenta samples were loaded and analyzed using R software (http://www.r-project.org/). Initially, we extracted the signal background of the slides and then filtered the probes that did not show fluorescence signal intensity. Data normalization was performed using the 90th percentile and subsequently transformed to a logarithmic scale. Highly expressed miRNAs in placenta was considered as the ones that presented expression (signal intensity) above the upper limit (outliers: points 1.5 IQR above the 3rd quartile) according the distribution of the microarray expression data in boxplot graphical tool. To the Cells were allowed to migrate/invade for 24 h at 37° C and 5% CO 2 and colony formation was evaluated after 12 days. CTRL: parental cancer cell line transfected with the irrelevant miRNA mimic (miRIDIAN mimic negative control); miR-451: cancer cell line transfected with miR-451 mimic; miR-720: cancer cell line transfected with miR-720 mimic. Vertical bars represent mean ± the standard deviation of independent triplicates. Mann-Whitney statistical test (migration and invasion); One-Way ANOVA statistical test (colony formation); * P < 0.05; ** P < 0.01; *** P < 0.001. www.oncotarget.com analysis and data visualization (charts and graphs), we used packages (limma and AgiMicroRNA) available at the public database Bioconductor (http://www.bioconductor.org). miRNA expression validation miRNA expression validation was performed for normal placenta and cancer cells lines using TaqMan MicroRNA Assays (Applied Biosystems, Foster City, CA, USA). Initially, 20 ng of total RNA was submitted to miRNA-specific cDNA synthesis using High Capacity Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) according to the following conditions: 1× enzyme buffer, 1 mM dNTPs, 1 µl of specific stemloop primer, 4 U RNAse inhibitor, 50 U Multiscribe Reverse Transcriptase in a final volume of 15 µl. Reaction conditions followed according to the manufacturer's instructions.
cDNA was used as template for the RT-qPCR using the TaqMan Fast Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) in the following conditions: 1X Master Mix, 0.5 µl of specific primer pair and probe, and 4 µl of cDNA (diluted in a ratio 1:4) to a final volume of 10 µl. All the reactions were performed in duplicates using standard reaction conditions in ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). miRNA endogenous controls (RNU24, RNU44 and RNU48) were also evaluated in all samples. Expression normalization was performed using RNU24, which presented the lowest expression variation (<1Ct) among all samples. Normalized expression levels of miRNAs were calculated using the mathematical model proposed by Livak and Schmittgen (2001) [47].

miRNA ectopic expression and functional assays miRNA mimics transfection
The ectopic expression of miR-451 or miR-720 was performed in cancer cell lines JEG3 (choriocarcinoma) and HT-29 (colon adenocarcinoma). miRNA overexpression was performed using miRIDIAN mimics assay (Dharmacon, Lafayette, CO, USA). Cell line transfections were carried out with DOTAP (Roche Applied Science, Indianapolis, IN, USA) following the protocol described by Martino et al. (2009) [48].

Migration and invasion assays
Cell motility and invasion ability were evaluated in 24-well transwell inserts (8 µm pores -Greiner Bio-One, Monroe, NC, USA) or 24-well transwell inserts containing Matrigel (Corning, New York, NY, USA), respectively. JEG3 and HT-29 cells (1 × 10 5 cells/200 µL serum free medium) were seeded in the upper chambers of the transwell inserts 48 h after transfection with mimics for miR-451 or miR-720 or negative control. Lower chambers were filled with 500 µL of medium added 10% FBS. JEG3 cells were allowed to migrate/invade for 24 h at 37° C and 5% CO 2 . HT-29 cells were allowed to migrate for 24h and invade for 48 h at 37° C and 5% CO 2 . Then, cells from the upper compartment were removed with a cotton swab and insert membrane were fixed with 4% formaldehyde (in PBS) and stained with 0.5% crystal violet. The number of cells was manually counted using ImageJ software. All experiment conditions were performed in triplicates.

Colony formation assay
Cells overexpressing miR-451 or miR-720 for 48h were harvested using TrypLE Express enzyme (ThermoFisher Scientific, Waltham, MA USA) and plated sparsely into 6-well plates (500 cells/well). After 12 days, cells were washed with PBS 1×, fixed in 4% formaldehyde (in PBS) and stained with 0.5% crystal violet. The number of colonies was counted using ImageJ software. All experiment conditions were performed in triplicates.

Author contributions
DOV and WAS designed the study. DOV and GAM collected samples and GD contributed with sample colletion. DOV processed samples for microarray assays, interpreted, analyzed and validated the results. DGP performed microarray data analysis. DOV, AR, BRM, JCCL, CPA, DZ performed functional in vitro assays. DOV, AR and WAS wrote the manuscript. All authors approved the final version of the manuscript.