Na,K-ATPase Isozymes in Colorectal Cancer and Liver Metastases

The goal of this study was to define Na,K-ATPase α and β subunit isoform expression and isozyme composition in colorectal cancer cells and liver metastases. The α1, α3, and β1 isoforms were the most highly expressed in tumor cells and metastases; in the plasma membrane of non-neoplastic cells and mainly in a cytoplasmic location in tumor cells. α1β1 and α3β1 isozymes found in tumor and metastatic cells exhibit the highest and lowest Na+ affinity respectively and the highest K+ affinity. Mesenchymal cell isozymes possess an intermediate Na+ affinity and a low K+ affinity. In cancer, these ions are likely to favor optimal conditions for the function of nuclear enzymes involved in mitosis, especially a high intra-nuclear K+ concentration. A major and striking finding of this study was that in liver, metastasized CRC cells express the α3β1 isozyme. Thus, the α3β1 isozyme could potentially serve as a novel exploratory biomarker of CRC metastatic cells in liver.


INTRODUCTION
Colorectal cancer (CRC) is one of the major causes of neoplasia-related morbidity and mortality, representing the second major cause of disease incidence among females and the third among males (Jemal et al., 2011). In the western world, CRC is the 4th leading cause of death (Ferlay et al., 2010). Metastatic CRC cells can invade, populate and flourish in a new niche and ultimately cause organ dysfunction and death. CRC spawns metastases in liver, lungs, bone marrow and brain (Chiang and Massague, 2008). However, it is the liver where CRC cells metastasize most frequently (Hess et al., 2006). The first line treatment for CRC involves surgery and adjuvant oxaliplatin based chemotherapy. A common side effect of this treatment strategy is oxaliplatin-induced peripheral neuropathy (Pachman et al., 2014).
Previous research from our group has led to the identification of several genes, which were shown to be significantly up-, or down-regulated in peripheral white cells (PWCs) of CRC patients, due to oxaliplatin-based chemotherapy (Morales et al., 2014). Interestingly, one of the differentially expressed genes was the isoform α3 of the Na,K-ATPase; mRNA levels of Na,K-ATPase α3 subunit were down-regulated 2.6-fold. Moreover, an alteration in the intracellular location of Na,K-ATPase α3 isoform has been reported in human CRC tumor cells vs. normal colon (Sakai et al., 2004). Additionally, other laboratories have shown differential expression in cells, altered subcellular localization and down regulation of the β subunit of the Na + /K + -ATPase in carcinoma cells (Rajasekaran et al., 1999(Rajasekaran et al., , 2001a(Rajasekaran et al., ,b, 2010. Na,K-ATPase is an integral protein in the plasma membrane of all animal cells that transports three sodium ions out and two potassium ions into the cell, against electrochemical gradient (Skou, 1957;Jorgensen et al., 2003). This activity is necessary for the regulation of the cellular ionic homeostasis and maintaining the electrochemical gradient required for ion channel function and secondary active transport (Mobasheri et al., 2000). Recently, additional functions for the Na,K-ATPase in the cell have been proposed, as a signal transducer and transcription activator (Aizman et al., 2001;Miyakawa-Naito et al., 2003;Harwood and Yaqoob, 2005;Yuan et al., 2005;Zhang et al., 2006) affecting cell proliferation (Abramowitz et al., 2003), cell motility (Barwe et al., 2005), and apoptosis (Wang and Yu, 2005). Besides this, the Na,K-ATPase is the receptor of cardiotonic glycosides. It is functionally composed of catalytic α (100-112 kDa) and regulatory β (45-55 kDa) subunit and an optional γ (6.5-10 kDa) subunit belonging to the FXYD family of proteins (Mercer et al., 1993).
The purpose of this study was to determine the cellular and subcellular localization of the α and β subunit isoforms of Na,K-ATPase in CRC and its liver metastasis using a panel of well-characterized isoform-specific antibodies. The primary hypothesis of this study was that metastatic cancer cells possess a unique expression phenotype of Na,K-ATPase isozymes, similar to that of CRC cells.

Tissue Samples
The Ethics Committee of the Universidad de La Laguna (ULL) and Ethical Committee of the Hospital Universitario Nuestra Señora de Candelaria (HUNSC) approved this study. All patients signed an informed-consent document for diagnosis and research on tissue specimen before being enrolled in the project. All the study subjects were treated with FOLFOX CT: day 1 oxaliplatin 100 mg/m 2 iv over 2 h; leucovorin calcium 400 mg/m 2 iv over 2 h; followed by 5-fluorouracil 400 mg/m 2 iv bolus and by 5fluorouracil 2400 mg/m 2 iv over 46 h; every 14 days. Paraffinembedded tissue samples and clinical data were obtained from 15 patients (7 males, 8 females) and 1 control male from the reference medical areas of HUNSC. Table 1 shows antibodies and references used in this study. Secondary antibodies used were goat anti-rabbit IgG or goat anti-mouse IgG. Biotinylated secondary antibody was used for immunohistochemistry (IHC), whereas secondary antibodies targeted with specific fluorochromes were used for immunofluorescence (IF).
following the procedure stated above but without incubating with primary antibody.

Double Immunofluorescence Simultaneous Staining
As with the IHC samples, tissue sections for IF staining were paraffin embedded. After deparaffinization, hydration and heatinduced epitope retrieval procedure (as described above for the IHC staining), slides were incubated with 5%BSA, 0.3%Triton-X-100 in TBS to block non-specific sites. Then tissue sections were incubated simultaneously with a mixture of two distinct primary antibodies (e.g., rabbit against human target 1 and mouse against human target 2) overnight at 4 • C. Slices were then incubated for 1 h at room temperature in dark with a mixture of two secondary antibodies (see Table 1) conjugated to two different fluorochromes (i.e., FITC-conjugated against rabbit-Sigma and DyLight R 650-conjugated against mouse-Abcam). Antibodies for IF were diluted in TBS, 1% bovine serum albumin (BSA), 0.1% Triton. Slides were mounted with ProLong R Diamond Anti-fade Mountant with DAPI (Molecular Probes by Life Technologies, #P36962, Eugene, Oregon, USA) to visualize cell nuclei. Slides were acquired and analyzed using Olympus confocal microscope (Olympus FV1000, Tokyo, Japan) and the software FV10-ASW1.3; Lasers: Diode 405 nm, Argon multiline 458/488/514, HeNe 633 nm. Images were acquired by sequential scan (first sequence Diode and HeNe, second sequence Argon) to avoid overlapping of channels. Image resolution 1024 × 1024. Objective lens: 60X/1.35 NA oil Plan-Apochromat. Negative control experiments were carried out by following the same immunohistochemical procedure but with the primary antibody omitted.

Image Analysis and Scoring
Samples were evaluated by two independent observers who were blinded to the clinical data. Scores were graded as absent (−), moderate (+) or strong (+ + +) for any specific kind of cell. These cut-offs were established by consensus of all investigators. For all tumors this grading was applied to three different patterns of Na,K-ATPase α and β subunit isoform staining in tumor cells: staining of the plasma membrane; staining of the nuclear envelope and staining of the cytoplasm.
Final results were computed as the product of staining intensities. In cases where scorings differed, the observers reevaluated samples to consensus. All samples were analyzed and scored.

Na,K-ATPase α1 and α3 Isoform Expression in CRC
In healthy colon tissue (Figures 1A,C), the α1 isoform was detected at the basolateral side of the plasma membrane of epithelial cells lining the colonic mucosae of Lieberkühn Crypts and in discrete stromal cells in the connective tissue surrounding the crypts. In turn, α1 isoform was mainly detected in a perinuclear location in tumor cells (Figures 1B,D). Mesenchymal cells from the stromal tissue surrounding the tumor also exhibited positive immunostaining (Figures 1B,D).
The Na,K-ATPase α3 isoform was detected on epithelial cells lining the colonic crypts and on cells from the lamina propria in healthy colon ( Figure 1E). The α3 isoform was mainly detected in or near the plasma membrane of epithelial cells and in the cytoplasm of positively stained cells in the stroma. In CRC tumor samples, the α3 isoform was mainly located in a peri-nuclear location in CRC tumor cells, while in the plasma membrane of these cells staining was negative ( Figure 1F). Stromal cells surrounding the tumor were also α3-positive. Immunolabeling for the α3 isoform was also detected in microvascular endothelial cells ( Figure 1H) and in cells from the inflammatory reaction associated with CRC within the stroma (Figure 1G), where the α3 isoform showed an intense and specific peri-nuclear labeling.

Na,K-ATPase β1 Isoform Expression in CRC
The β1 isoform of Na,K-ATPase was detected in epithelial cells from the normal colonic mucosae (Figure 2A). There was a high positive staining at the baso-lateral side of polarized epithelial cells that line the colonic Lieberkühn crypts ( Figure 2B).
In CRC tumors, the β1 isoform presented a less defined expression pattern. This isoform was detected in some tumor cells, but the location was not well-defined, and was seen in several subcellular locations within the tumor. In some tumor cells β1 staining was peri-nuclear ( Figure 2C), while in others a peripheral location was observed ( Figure 2C).
In addition, in healthy tissue, β1 was detected in cells of the myenteric plexus, also known as the Auerbach's plexus, within the muscular tissue of the muscularis propia ( Figure 2E). In the cells from the plexus, Na,K-ATPase β1 isoform was detected in or near the cytoplasmic membrane of axons and dendrites of neurons and glial cells ( Figure 2F).

Na,K-ATPase β2 Isoform Expression in CRC
The β2 isoform was detected at the baso-lateral side of polarized epithelial cells from the normal colonic mucosae and in FIGURE 2 | Immunolocalization of the Na,K-ATPase β1 and β2 isoforms in normal colon and CRC. (A,B) β1 is mainly located at the baso-lateral side of polarized epithelial cells from the normal colonic mucosae (arrows in B). (C) β1 is located in the cytosol of tumor cells (arrows). (D) β1 is located at the cell membrane of tumor cells (arrows). (E) Cells from the myenteric plexus (MyP) at the muscularis propia (mp) express β1. (F) β1 isoform is mainly located in or near the cytoplasmic membrane of the neurons and/or glia; a neuron nucleus with visible nucleolus is indicated using an arrow. g, colonic gland (Lieberkühn crypts); lm: lamina propia mucosae. (G) Selected cells from the connective tissue of the mucosae express the β2 isoform (arrows). (H) β2 is mainly located at the baso-lateral side of polarized epithelial cells from the normal colon mucosae (arrows). (I) β2 was not detected in tumor cells from adenocarcinomatous glands (T); however, some stroma (S) cells express β2 (arrows). (J) Possible leukocytes of the stroma expressing β2 (arrows). (K) Cells from the myenteric plexus (MyP) at the muscularis propia (mp) express β2. (L) β2 isoform is mainly located at the soma of glia cells (arrows).
Frontiers in Physiology | www.frontiersin.org selected fibroblastic and immune cells from the lamina propria ( Figure 2G) and (Figure 2H). In colon adenocarcinoma cells, the β2 isoform was not detected (Figures 2I,J). However, some immune cells located in the stromal tissue surrounding the tumor, were β2 positive while others remained negative (Figures 2I,J). In immunopositive cells, β2 staining was detected peri-nuclearly and also throughout the cytoplasm. Immunostaining intensity for the β2 isoform varied from strong to weak across cells in this region. In myenteric plexus ( Figure 2K) the β2 isoform was detected in the soma of neural cells (Figure 2L).

Na,K-ATPase α1 and α3 Isoform Expression in CRC Metastases in Liver
In healthy liver tissue, hepatocytes were immunopositive for α1, in the plasma membrane ( Figure 4A). Bile ducts cells were also α1 positive (Figure 4B), and the staining was detected mainly at the baso-lateral side of the plasma membrane. In metastases, the Na,K-ATPase α1 isoform was detected in cytoplasm and in the cytoplasmic membrane of cells of metastatic tumor niches (Figures 4C,D).
In normal healthy liver, Na,K-ATPase α3 isoform was not detected in any cell types ( Figure 4E). However, in metastatic tumor cells within the liver, the α3 isoform was detected ( Figure 4F; Supplementary Figure S1D) in a peri-nuclear location and spread across the cytoplasm. Staining intensity varied among cells, from strong to weak labeling. In addition to tumor cells, this isoform was also detected in immune cells located at the outermost part of the liver (Figure 4G). In apparently healthy liver tissue surrounding metastases, Na,K-ATPase α3 isoform was detected peri-nuclearly and also throughout the cytoplasm of hepatocytes ( Figure 4H).

Na,K-ATPase β1 and β2 Isoforms Expression in Metastasis
In metastasized liver, the β1 isoform was detected at the plasma membrane of hepatocytes (Figure 5A), bile ducts epithelial cells ( Figure 5B) and peri-nuclearly and/or in the cytoplasm of cells in disorganized and necrotic tissue (Figures 5C,D; Supplementary Figure S1C).
In healthy liver tissue, the β2 isoform was detected in the cytoplasm of some cells and in peri-nuclear locations in others (Figures 5E,F), with variable staining intensities among cells ranging from strong to weak. A weak but specific signal for the β2 isoform was also detected in bile ducts cells at the portal triads ( Figure 5G). However, the β2 isoform was not detected in metastatic tumor cell niches ( Figure 5H).
We were unable to detect the α2 and β3 subunit isoforms as their expression levels were probably below the threshold of the ABC amplified immunohistochemical detection technique employed in this study.

Na,K-ATPase α3 and β1 Isoforms Coexpression in Metastasis
In order to further confirm the co-expression of the α3 and β1 subunits isoforms in the same metastatic cells in the liver, using a different and monoclonal antibody, we performed confocal microscopy co-localization experiments. Image analysis and scoring was done using same procedure as in all other cases  (E) Negative immunolocalization of the α3 isoform in normal liver tissue. (F) α3 isoform is mainly located at a peri-nuclear position but also detected all-over the cytoplasm. (G) Inflammatory reaction established at the outermost part of the liver; α3 IHC-positive cells may correspond to cells from the immune system (arrows), such as leukocytes. (H) In normal liver tissue from a metastasized liver, hepatocytes express α3 in peri-nuclear and cytoplasmic locations. and stated in the Materials and Methods section. As shown in Figure 6 and in Supplementary Figure S1, both of them colocalize in a number of metastatic cells in percentages ranging from + to +++, depending on the sample and on the area within the same sample. Most of them showed further more than 2/3 of total metastatic cells.

DISCUSSION
In this study we explored the cellular and subcellular localization of the α and β subunit isoforms of Na,K-ATPase in CRC and its liver metastases. The aim of this work was to test the hypothesis that metastatic cancer cells possess a unique expression phenotype of Na,K-ATPase isozymes, which may be similar to that of CRC cells. Table 2 summarizes the cellspecific Na,K-ATPase subunit-isoforms expression and Table 3 highlights the possible cell-specific Na,K-ATPase isozymes present in healthy colon, colorectal cancer, healthy liver and metastasized liver.
Based on the results presented we propose that the predominating isozymes in tumor cells from the colon and metastases in the liver are α1β1 and α3β1. The α3β1 isozyme of Na,K-ATPase is only present in liver metastases but not in healthy liver, thus, the α3β1 isozyme could serve as a novel exploratory biomarker of CRC metastatic cells in liver. Further studies should be carried out to test the utility of this observation.
Subunits of Na,K-ATPase have the ability to form functional isoenzymes by a promiscuous association of α and β isoforms  Endothelial cells ?

Metastasized liver
Tumor cells + + + + + + + − to confer significantly different kinetic and biological properties. The apparent affinities for cations and ouabain have been determined by expressing recombinant enzymes in heterologous systems. Affinities of human isozymes expressed in Xenopus laevis oocytes are α1β1>α2β1>α3β1 for Na + and α3β1=α1β1>α1β3>α1β2>α2β1>α3β3>α3β2>α2β3>α2β2 for K + (Blanco and Mercer, 1998;Crambert et al., 2000). Tumor cells and metastatic cells have isozymes with highest and lowest Na + affinity and surrounding mesenchymal cells possess isozymes with medium range affinity. Regarding K + affinity, tumor and metastasis cells possess Na,K-ATPase isozymes of high K + affinity and mesenchymal cells low K + affinity. These are the isozyme combinations that permit an optimal performance of the enzymes involved in protein synthesis and transfer of phosphor groups (Glynn, 1985) processes both involved in carcinogenesis.

Hepatocytes
Another objective of this study was to correlate the mitotic index related to the expression of isoforms by co-localization of those along with PCNA, the clamp subunit of DNA polymerase δ marker of cell proliferation (Kubben et al., 1994;Bleau et al., 2014) and carried out further analysis by confocal microscopy. Figure 3 shows cells expressing the α3 and β1 isoforms in CRC tumor cells, in which no correlation was seen between sodium pump isoforms and PCNA protein expression, that is, high 3 | Possible cell-specific Na, K-ATPase isozymes present in normal colon, colorectal cancer, normal liver, and metastasized liver. expression of PCNA can be found in cells with either, high or low, expression level of α3 or β1 and vice versa. Hideki Sakai and co-workers (Sakai et al., 2004) used western blotting to demonstrate a decrease in α1 isoform expression in CRC and, inversely, an increase in the α3 isoform compared to the accompanying healthy mucosae. In addition they did not observe a significant expression level of Na,K-ATPase α2 isoform either in the CRC or in the accompanying healthy mucosae. Our study, confirms their observations. In addition, in recent studies of hepatocellular carcinoma, a significantly higher α3 level expression was shown in western blots compared to the accompanying non-tumor tissues, whereas no significant increases in expression of α1 and α2 proteins was observed (Shibuya et al., 2010).
Recently, it has been suggested that the cellular distribution and expression of Na,K-ATPase α3 isoform affects the antiproliferative effects of oleandrin, a cardiac glycoside that inhibits the Na,K-ATPase (Yang et al., 2014). These authors demonstrated that healthy, as opposed to neoplastic colonic and lung tissues, exhibit different distributions of the α3 isoform. While the α3 isoform was predominantly located in the cytoplasmic membrane in healthy colon and lung, the distribution of this isoform was shifted to a predominantly peri-nuclear location in tumors. These observations have been corroborated by our laboratory. Furthermore, our results showed a subcellular location shift for the α1 isoform, which was mainly located at the basolateral side of the plasma membrane of healthy colonic epithelial cells, shifting to a peri-nuclear position in CRC tumor cells.
The α1 and α3 subunit isoforms were detected in all cells lining the colonic crypts. These isoforms were not only expressed in epithelial cells in healthy colon mucosae, but they were also detected in mesenchymal cells from the lamina propria. The α3 isoform presents a high expression level in neurons of the central nervous system (Hieber et al., 1991;McGrail et al., 1991). The present study shows specific staining for α3 in neurons from myenteric plexus.
Regarding the β subunit, it has been reported that expression of both the β1 and β2 mRNAs were decreased in renal, lung and hepatocellular carcinomas (Akopyanz et al., 1991), and that expression levels of the corresponding proteins was decreased in human clear cell renal cell carcinoma (Rajasekaran et al., 1999) and bladder carcinoma (Espineda et al., 2004). Previous work from our laboratory (Avila et al., 1997) reported, by western blot technique, that gastric and colon adenocarcinoma showed opposite patterns of β1 isoform expression. While gastric adenocarcinomas showed lower expression levels of β1 than the healthy tissue, colonic adenocarcinomas showed higher expression of this isoform compared to healthy surrounding tissue. In addition, the β2 isoform was neither detected in healthy colon, nor in stomach adenocarcinomas. In the present immunohistochemical study, we detected the β1 and β2 isoforms at the baso-lateral side of the plasma membrane in the healthy colon mucosae, but only β1 was found in CRC samples. Which, in a certain manner, resembles our previous findings in Na,K-ATPase in dog and rat prostate cancer where we found a downregulation and a reduced expression of sodium pump (Mobasheri et al., 2000(Mobasheri et al., , 2003a. Na,K-ATPase β1 and β2 isoforms were detected in the myenteric plexus of healthy colon tissue. It is well-established that the β2 isoform of Na,K-ATPase, is an adhesion molecule on glia (AMOG) (Antonicek et al., 1987;Gloor et al., 1990).
In tumor samples, the β1 isoform presented a less defined pattern of expression. This isoform was detected in some tumor cells but not all, also the subcellular location differed among cells within a given adenocarcinomatous area, while some tumor cells where immunopositive for β1 at the cytoplasmic membrane location ( Figure 2D) other cells presented immunostaining in a peri-nuclear position ( Figure 2C).
Research by Rajasekaran and colleagues reported that Na,K-ATPase β subunit is required for epithelial polarization, suppression of invasion, and cell motility (Rajasekaran et al., 2001b), not only presence of Na,K-ATPase in the cell membrane but also Na,K-ATPase activity was important to form proper tight junctions, desmosomes, and induction of polarity in epithelial cells (Rajasekaran et al., 2001b). Further studies suggested that the transcription factor Snail might be repressing the β1 isoform and E-cadherin expression in carcinomas, associating these events to epithelial-mesenchymal transition (EMT) (Espineda et al., 2004).
Taken together, these studies and our results indicate that the level of expression and the location of the β subunit in epithelial cells are important for maintaining their well-differentiated phenotype, which disappears during cancer progression. Research published by our group on sodium pump isoform expression levels in stem cells has confirmed that adipose-derived mesenchymal stem cells express all known Na,K-ATPase isoforms, but some of these genes are turned off along differentiation (Acosta et al., 2011). In CRC cells or its metastases in liver we have never seen expression of all isoforms, rather, we have detected the expression signatures specified in Table 2.
Regarding liver metastases, to our knowledge, there have not been any reported studies in reference to Na,K-ATPase isoforms in liver metastasis. In this study the α1 isoform was detected in metastatic tumor cell niches within the liver, exhibiting a cytoplasmic subcellular localization in some cells and a membrane localization in others. The α3 isoform, however, was mainly detected at a peri-nuclear location and was more diffusely expressed across the cytoplasm of tumor metastatic cells. The healthy hepatic tissue presented the α1 isoform at the cytoplasmic membrane where it may establish the functional heterodimer with a β1 isoform and/or β2 isoform. Our observations of the subcellular localization of the α1 isoform are consistent with the first reported immunolocalization of this subunit in hepatocytes of healthy liver tissue (Sztul et al., 1987). However, in healthy liver tissue, the α3 isoform was not detected.
The β2 isoform was detected both at a more peri-nuclear position in some hepatocytes and throughout the cytoplasm in others, but not at the cytoplasmic membrane. The reason for this is unclear at present. It is possible that β2 isoform performs other moonlighting protein functions in hepatocytes. In metastasized liver, we detected the β1 isoform in disordered and semi-necrotic tumor tissue. However, β2 isoform was not detected in liver metastases. This may be related to the fact that these metastatic cells arise from CRC tumor cells, which did neither express β2 isoform or at very insignificant levels. Interestingly, apparently in healthy hepatic tissue surrounding the metastatic zone, the hepatocytes expressed the α3 isoform, a phenotype not detected in healthy hepatocytes from non-CRC patient according to our results. It might be possible that the CRC and the FOLFOX-CT affecting this patient could be influencing these hepatocytes driving them to express other genes, ATP1A3 in this case.
The high levels of peri-nuclear and cytoplasmic α3 isoform in liver metastatic cells is potentially indicative of other moonlighting functions of this isoform besides ion transport (Jeffery, 2014;Magpusao et al., 2015;Min et al., 2015). In addition, the α3β1 isozyme may have utility as a novel exploratory biomarker for metastases cells. However, further studies need to be performed in order to confirm both, the moonlighting and biomarker assessments.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fphys. 2016.00009