Research ArticleExperimental Studies

Effects of SRC and STAT3 upon Gap Junctional, Intercellular Communication in Lung Cancer Lines

MULU GELETU, STEPHANIE GUY and LEDA RAPTIS
Anticancer Research October 2013, 33 (10) 4401-4410;

Abstract

Background: We have previously demonstrated a positive correlation between SRC and its effector signal transducer and activator of transcription-3 (STAT3), and a reverse relation between SRC and gap junctional communication (GJIC) in seven non-small cell lung cancer (NSCLC) lines. Since a number of oncogenes besides SRC can affect GJIC, here we examined the actual contribution of the SRC/STAT3 axis to GJIC suppression. Materials and Methods: SRC and STAT3 activity levels were examined in SK-LuCi-6, LC-T, QU-DB, SW-1573, BH-E, Calu-6, FR-E, SK-MES, H1299, BEN, WT-E, A549 and SHP-77 cells by western blott analysis, probing with antibodies specific for SRC-ptyr418 or STAT3-ptyr705. GJIC was examined by in situ electroporation. Results: Confluence of all cultured NSCLC cells tested induces a dramatic increase in STAT3 activity, which is independent of SRC action. In addition, the LC-T line had high STAT3-705, despite the fact that SRC-418 expression was low, indicating that other, SRC-independent factors must be responsible for STAT3 activation and GJIC suppression in these cells; however, BH-E and SHP-77 cells with low GJIC, both SRC-418 and STAT3-705 expression were low, indicating that GJIC suppression can be independent of the SRC/STAT3 axis altogether. Our results also show that STAT3 inhibition does not restore GJIC in any of the examined lines, while in the non-transformed rat F111 fibroblast line which has extensive GJIC, STAT3 inhibition actually eliminated junctional permeability. Conclusion: Our results indicate a further level of complexity in the relationship between SRC, STAT3 and GJIC in NSCLC than what has been previously demonstrated. In addition, STAT3 is actually required for, rather than suppressing GJIC.

Gap junctions are channels crossing the plasma membrane of cells. They connect the interiors of neighboring cells and are formed by the connexin (CX) family of proteins. Oncogene proteins with tyrosine kinase activity such as SRC may phosphorylate the ubiquitous gap junction protein, CX43, directly or indirectly, thereby reducing gap junctional, intercellular communication (GJIC) (1).

Examination of tyr-418 phosphorylation of SRC (SRC-418), which correlates with its activity, in a number of non-small cell lung cancer (NSCLC) biopsies revealed higher SRC activity than the surrounding, normal lung tissue (2, 3). However, the contribution of SRC itself to GJIC suppression in NSCLC lines which may express other oncogenes in addition to SRC, remains to be determined.

SRC has a number of downstream effector pathways, such as the RAS/RAF/ERK, phosphatidyl-inositol-3 kinase (PI3K)/AKT, CRK-associated substrate (CAS), the signal transducer and activator of transcription-3 (STAT3) and others (4). Although SRC is known to phosphorylate CX43 on tyr265 and tyr247 directly, whether it is this phosphorylation which inhibits GJIC or whether some of the SRC effector pathways, which can suppress GJIC on their own, play a more important role is still controversial (1). STAT3 is a cytoplasmic transcription factor that, following phosphorylation on tyr-705 by SRC, as well as by growth factor or cytokine receptors such as the interleukin-6 (IL6) family, normally dimerises through a reciprocal interaction between the phosphotyrosine and the SH2 domain and translocates to the nucleus, where it induces the transcription of specific genes (5). Previous findings showed that SRC is an important STAT3 activator in certain NSCLC lines (6), but in a later report SRC inhibition in different NSCLC lines was found to actually increase STAT3 activity (7). We and others recently demonstrated that cadherin engagement, as occurs in confluent, cultured cells, causes a dramatic increase in STAT3 activity [reviewed in (8)]; therefore, cell density effects might account for these apparent discrepancies. In the present communication we revisited the question of the relationship between SRC and STAT3 in NSCLC, by assessing STAT3 activity at a range of densities, through measurement of STAT3, tyr-705 phosphorylation (STAT3-705), which correlates with its activity.

Examination of the mechanism of the SRC-mediated, GJIC suppression indicated that inhibition of the RAS (9) or CAS (10) pathways in SRC-transformed, rat fibroblasts reinstated GJIC. Conversely, expression of the middle tumor antigen of polyoma virus (mT) which acts by activating cSRC, in RAS-deficient cells was unable to suppress GJIC (11). Taken together, these data indicate that the RAS pathway is involved in the SRC-initiated GJIC suppression. However, the effect of the SRC/STAT3 axis upon GJIC in NSCLC is unclear. Using a novel technique of in situ electroporation on a partly conductive slide (12), we recently demonstrated that NSCLC lines A549, SK-Lu1, CALU-1, SW-900, CALU-6, with high SRC-418 levels had low GJIC, while lines QU-DB and SK-LuCi6 with low SRC-418 had high GJIC (12), consistent with the documented ability of SRC for GJIC suppression. Here, we extended this study to a further six lines (SW-1573, WT-E, BEN, H1299, FR-E, SK-MES), and these results reveal a further level of complexity. We also showed that STAT3 inhibition in non-transformed lines with extensive GJIC eliminated junctional permeability, indicating that, rather than suppressing GJIC, STAT3 is actually required for GJIC.

Materials and Methods

Examination of GJIC. The technique previously described (12, 13) was employed, using the ACE-100, InSitu Porator apparatus and ACE-08-CC cuvettes, available from Cell Projects Ltd, 2 Roebuck Business Park, Ashford Road - Harrietsham -Kent ME17 1AB, UK, ph: 4400 1622 851177, www.cellprojects.com.

Briefly, cells were grown on electroporation chambers, the bottom of which consisted of a glass slide, coated in part with optically-transparent and electrically-conductive, Indium-Tin oxide (ITO). At 3 days after confluence, the growth medium was replaced with Calcium-free DMEM supplemented with 5 mg/ml Lucifer yellow (LY). A set of electrical pulses (10 alternating pulse pairs of 18 Volts) which open transient pores on the cell membrane, was delivered to the cells growing on the ITO-coated, i.e. conductive part of the slide. Following a 5 min incubation in a humidified, CO2, 37°C incubator for the transfer of LY to the neighboring, non-electroporated cells to occur, the unincorporated dye was washed away with Calcium-free DMEM supplemented with 10% dialysed fetal calf serum. The migration of the dye from the electroporated cells growing on the coated part of the slide, to the neighboring, non-electroporated cells was then microscopically observed and the cells photographed, under fluorescence and phase contrast illumination. Communication was expressed as the number of cells into which the dye has transferred, per cell loaded with the dye by electroporation at the edge of the electroporated area. All experiments were conducted at least three times, with at least 5 slides each time, and the results are presented as average GJIC±SEM where the transfer from at least 200 cells is assessed.

Cell lines, culture techniques and STAT3 activity measurement. Cell lines CALU-6, SW-1573, WT-E, FR-E, SK-MES, LC-T, BH-E, QU-DB and SK-LuCi6 have been described previously (14). SHP-77 is an undifferentiated, large cell variant of a small cell lung carcinoma line and was obtained from the American type culture collection (ATCC) [Catalog number: ATCC 30-2001 (15)]. BEN cells were obtained from ATCC [Catalog number: ATCC ACC 254 (16)]. H1299 cells (ATCC CRL-5803) were a gift from Dr. Xiaolong Yang, Queen's University. All cells were grown in Dulbecco's modification of Eagle's medium (DMEM) with 10% fetal calf serum (14). Care was taken to ensure that cell seeding was uniform by passing cells at sub-confluence, when cell to cell adhesion was low. Confluence was estimated visually and quantitated by imaging analysis of live cells under phase contrast microscopy (17). Cells were pelletted by centrifugation prior to extraction, in order to avoid problems in protein determination due to serum proteins attaching to the plastic tissue-culture dish (18).

Inhibitors. STAT3 was inactivated using two approaches: Treatment with 50 μM PtCl3(NO2)(NH3)2 (CPA7), prepared as we described before (19) for 24 h, or with 100 μM S3I-201 (a gift of Dr. Turkson, University of Hawaii) (20), for 24 h. Both compounds were prepared in 50% dimethylsulfoxide (DMSO) as 200-times concentrated stocks and added to the growth medium.

SRC was inactivated using the pharmacological inhibitors dasatinib (0.5 or 1 μM for 24 h) or PD180970 (0.2 μM with redosing every 12 h for a total of 24 h), (21), both gifts from Dr. Turkson, or an Adenovirus vector expressing a dominant-negative SRC mutant [a gift from Dr. Kaplan, University of Toronto (21)]. Although at concentrations of 0.1-1 μM dasatinib eliminated SRC-418, total SRC was found to be slightly increased in certain experiments.

Western blotting. Werstern blots were conducted on proteins extracted from cell pellets (18), following the indicated treatments. The following antibodies were used: CX43 (Cell Signalling, #3512, used at a 1:500 dilution); STAT3-ptyr705 (Cell Signalling, #9131, 1:1,000); SRC-ptyr418 (Invitrogen, #44-660G, 1:1,000); or total SRC (rabbit monoclonal 36D10, Cell Signalling, #2109, 1:1,000), followed by secondary antibodies and ECL reagents (Biosource). Alpha tubulin (Cell Signalling #2125, 1:5,000); GAPDH (BD Transduction, #14C10, 1:5,000); or HSP90 (Assay Designs, #SPA-830, 1:5,000) served as loading controls. Quantitation was achieved by scanning the autoradiograms after multiple exposures.

Results

Density-induced, STAT3 activation is independent of SRC. To examine the effect of SRC upon GJIC, it is important to assess the effect of cell density, which is a prerequisite for the assembly of adherence junctions and GJIC (22, 23), upon SRC activity. In fact, it has been reported that cell-to-cell adhesion may dramatically increase the activity of the prominent SRC effector, STAT3 [(17, 21, 24-26), reviewed in (8)]. Therefore, to examine the effect of density upon SRC-418, we expressed activated SRC in the lung cancer line SK-LuCi6, previously shown to have low SRC-418 expression and extensive GJIC (12), with a retroviral vector (line SK-LuCi6-Src) (27). SK-LuCi6 and SK-LuCi6-Src cells were grown to progressively increasing densities from 80% of confluence up to three days post-confluence, that is the densities where STAT3-705 phosphorylation and activity were previously shown to be reaching their highest levels in a large number of lines (8, 17) and detergent cell extracts were subsequently probed for SRC-418 and STAT3-705. As shown in Figure 1, cell density had no effect upon SRC-418 or total SRC protein levels in either line, while as expected, SRC levels were higher in SK-LuCi6-Src cells. At the same time, STAT3-705 levels dramatically increased with increasing density in SK-LuCi6 cells (Figure 1B), and were certainly higher in SK-LuCi6-Src than SK-LuCi6 cells at all densities examined. These findings indicate that density per se does not affect SRC levels or activity in these cells, consistent with previous data obtained rodent fibroblasts (28), and point to the possibility that SRC may not be the kinase responsible for the STAT3-705 increase observed at high cell densities.

To further examine the role of SRC upon STAT3-705 in NSCLC, we tested the effect of the SRC inhibitor, dasatinib on SK-LuCi6-Src cells. As shown in Figure 1, dasatinib treatment caused a dramatic reduction in SRC-418, while total SRC levels remained unaffected. Interestingly, SRC inhibition at low densities essentially eliminated STAT3-705 expression, indicating that SRC is the main contributor to STAT3-705 phosphorylation. Most importantly however, dasatinib treatment of SK-LuCi6-Src cells at two days post-confluence, caused only a small reduction in STAT3-705 levels (compare lanes 3 vs. 5, Figure 1A). Furthermore, dasatinib treatment of SK-LuCi6 cells did not reduce STAT3-705 in cells grown to high densities (Figure 1B, lanes 2 vs. 4). Similar results were obtained with the inhibitor PD180970 and through infection with an adenoviral vector expressing a dominant-negative SRC mutant, as before (21), and with the mouse lung epithelial line E10 (29) before and after SRC expression (data not shown). Taken together, these data indicate that the density-induced STAT3 activation is independent of SRC in NSCLC, consistent with data from mouse fibroblasts and epithelial cells (21, 30). Therefore, to avoid the confounding factor of density, the effect of SRC upon STAT3-705 was examined at 50% confluence in all subsequent experiments.

SRC as a STAT3 activator in NSCLC lines. Besides SRC, STAT3 is known to be a prominent effector of other cytokine and membrane tyrosine kinase receptors, which may be present in an activated form in NSCLC cells and up-regulate STAT3 activity. Therefore, to examine the effect of SRC specifically upon STAT3-705, we examined the correlation between SRC-418 and STAT3-705 levels in a number of NSCLC lines. As shown in Figure 2 (A and B), Calu-6, SW-1573, WT-E, BEN, H1299, FRE and SK-MES cells displayed high levels of SRC-418, approximately 95% to 25% that of SK-LuCi6-Src (or E10-Src, Figure 2A, lane 9) and these levels were found to correlate with their levels of STAT3-705 at low cell densities. These data point to the possibility that SRC may be a significant contributor to STAT3 activity in these lines. In sharp contrast however, the LC-T cell line displayed high STAT3-705 expression despite the fact that SRC-418 was not detectable (Figure 2A, lane 2). Similarly, SK-MES cells had intermediate SRC-418 levels (25% that of SK-LuCi6-Src), although STAT3-705 expression was 91% that of SK-LuCi6-Src, and FR-E cells with SRC-418 expression of 25% displayed STAT3-705 expression of 70% that of SK-LuCi6-Src (Figure 2 B), indicating that other SRC-independent factor(s) must also be responsible for the high STAT3-ptyr705 expression in these lines. The levels of total STAT3 differed from line to line, although the observed differences were not as pronounced as the levels of active STAT3-705 (Figure 2D). This could be due to the fact that STAT3 activates its own promotor (31).

Figure 1.
Figure 1.

Density-induced, STAT3 activation is independent of SRC. A. SK-LuCi6 (lane 1) or SK-LuCi6-Src (lanes 2-5) cells were grown to increasing densities as indicated and treated with dasatinib (lanes 4 and 5) or not (lanes 1-3). Detergent cell extracts were probed for SRC-418, STAT3-705, total SRC or tubulin as a loading control. Numbers at the left refer to molecular weight markers. Numbers under the lanes refer to relative band intensities obtained after quantitation, with the levels of SK-Luci6-SRC cells at three days post-confluence taken as 100%. B. SK-LuCi6 cells were grown to 80% confluence or 2 days post-confluence and treated with dasatinib (lanes 3 and 4) or not (lanes 1 and 2). Detergent cell extracts were probed for SRC-418, STAT3-705, total SRC and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control, as indicated. Numbers under the lanes refer to relative band intensities obtained after quantitation, with the levels in untreated cells grown to two days after confluence taken as 100%. Note that dasatinib was unable to reduce STAT3-705 expression in cells grown to two days after confluence.

Figure 2.
Figure 2.

SRC-418 and STAT3-705 expression in NSCLC lines. A and B: The indicated NSCLC lines were grown to 50% confluence and extracts were probed for SRC-418, total SRC, STAT3-705 and GAPDH as a loading control. Numbers at the left refer to molecular weight markers. C. Cell density-induced STAT3 activation is independent of SRC expression in SW-1573 cells. Cells were grown to increasing densities and treated with dasatinib, or not. Detergent cell extracts were probed for SRC-418, total SRC, STAT3-705 and GAPDH as a loading control. Numbers at the left refer to molecular weight markers. Numbers under the lanes refer to relative band intensities obtained after quantitation, with the levels of SK-Luci6-Src cells taken as 100%. D. The same extracts as shown in A and B were probed for total STAT3.

Figure 3.
Figure 3.

STAT3 inhibition reduces GJIC. A: Rat F111 fibroblasts were plated in electroporation chambers (see Materials and Methods) and at two days post-confluence treated with the the DMSO carrier alone (a-c) or with the STAT3 inhibitor S3I-201 (d-f). Lucifer yellow was electroporated and cells photographed under phase-contrast (a, d) or fluorescence (b, e) illumination. c, f: Merged phase-contrast and fluorescence photographs. Note the absence of transfer following STAT3 inhibition (e). Magnification: x200. B: SW-1573 cells were electroporated, as above, following treatment with the DMSO carrier (a, b) or the STAT3 inhibitor S3I-201 (c, d). Magnification: x140. C: Calu-6 cells were treated with the STAT3 inhibitor S3I-201 or the DMSO carrier and detergent cell extracts probed for STAT3-705 and SRC-418, as indicated. Note the dramatic reduction in STAT3-705 expression with the use of S3I-201.

Figure 4.
Figure 4.

Schematic of STAT3 activity levels as a function of cell density and SRC activity in cells with high SRC-418 (A) vs. low SRC-418 expression (B). In (B), the confluence-mediated STAT3 activation is resistant to SRC inhibition. In cells with high SRC-418 expression however (A), there are two pathways contributing to STAT3 activation: An SRC-dependent pathway, which is the same at all densities, and a cadherin-mediated pathway, which is independent of SRC and increases dramatically with confluence. Notably in this case, since cadherins may be partially degraded by SRC expression, the increase due to cell confluence may be lower than in cells with low SRC expression.

We next assessed the actual contribution of SRC to STAT3-705 levels directly in SW-1573 cells by assessing the ability of dasatinib to reduce STAT3-705 expression, in different cell densities. As shown in Figure 2C (lanes 1-3), cell density had no effect upon SRC-418 in SW-1573 cells, while dasatinib treatment caused a dramatic reduction in SRC-418 levels at all cell densities examined (lanes 4-6). However, examination of STAT3-705 levels indicated that although in sparsely growing cells dasatinib caused a dramatic reduction in STAT3-705 expression (lane 1 vs. 4 and 2 vs. 5), in confluent cultures the reduction was only ~50% (lane 3 vs. 6), consistent with results for the SK-LuCi6-Src cells (Figure 1). Similar results were obtained with the inhibitor PD180970 (21) and with Calu6, WT-E, BEN and H1299 cells (Table II). These findings further confirm that while in sparsely-growing cells highly expressing SRC-418, SRC is a major STAT3 activator, cell density-induced STAT3 activation is independent of SRC. At the same time, in cells with low SRC (e.g. BHE and SHP-77, Figure 2 and Table I), the increase in STAT3-705 expression observed at high confluence was resistant to dasatinib treatment (Table II), in agreement with data from SK-LuCi6 cells (Figure 1B). Taken together, these findings further reinforce the observation that cell density-dependent STAT3-tyr705 phosphorylation is also independent of SRC in these NSCLC lines.

Effect of SRC and STAT3 upon GJIC in NSCLC lines. Recent results demonstrated an inverse relationship between SRC and GJIC in certain NSCLC lines; five lines with high SRC-418 expression (A549, SK-Lu-1, CALU-1,SW-900, CALU-6) had low or undetectable gap junctional permeability while two lines with very low SRC levels (QU-DB, SK-LuCi6) had extensive GJIC (12), consistent with the established role of SRC as a GJIC suppressor. This suggestion was confirmed here with lines SW-1573, WT-E, BEN, H1299, FR-E and SK-MES, which have high SRC-418 expression (Table I). To explore the possibility that other oncogenes might also be involved in GJIC suppression in NSCLC, we assessed GJIC levels in lines with low SRC-418 expression, namely LC-T, BH-E and SHP-77 (Figure 2 and Table I). Cells were plated in electroporation chambers, Lucifer yellow introduced with a pulse to cells growing on the conductive part of the slide and the movement of the dye through gap junctions was observed under fluorescence illumination (see Materials and Methods). The results revealed that these cells have very low GJIC as well (Table I), despite the fact that SRC-418 expression was undetectable, indicating that other SRC-independent factors may play a role in reducing gap junctional permeability in these lines.

The role of STAT3, the prominent effector of a number of tyrosine kinase oncogenes known to suppress GJIC was examined next. STAT3 activity was repressed with the pharmacological inhibitor S3I-201 (20) in the lines with high SRC-418 expression (Calu6, SW-1573, WT-E, BEN, H1299, FRE, SK-MES), and GJIC quantitated. The results showed that this treatment, which effectively eliminated STAT3-705 expression, did not increase GJIC in SW-1573 cells (Figure 3 and Table I), consistent with previous data (12). We then extended these observations to LC-T cells which display high STAT3-705 expression despite the fact that expression of SRC-418 is low. Although S3I-201 caused a dramatic reduction in STAT3-705 expression in LC-T cells, it did not increase GJIC (Table I), indicating that STAT3, which is likely activated by an SRC-independent mechanism, may not be part of a pathway leading to gap junction closure in these cells. Similarly, the STAT3 inhibitor CPA7 (32) did not increase GJIC in LC-T cells (data not shown). Taken together, these data reinforce the conclusion that STAT3 is not a factor of GJIC suppression by SRC or other oncogenes.

Figure 5.
Figure 5.

RAC1 down-regulation reduces STAT3-tyr705 phosphorylation in SK-LuCi6 cells. shRAC was expressed in SK-LuCi6 cells with a retroviral vector (34), packaged with an amphotropic line (12). Extracts from stably-expressing cells grown to densities of two days post-confluence were probed for RAC1, phosphorylated STAT3-tyr705 and GAPDH as a loading control, as indicated. Note the reduction in phosphorylated STAT3-tyr705 levels upon RAC1 down-regulation. The residual phosphorylated STAT3-tyr705 levels may be due to STAT3 activation by Cdc42 (30, 34).

We next examined the effect of STAT3 inhibition in BH-E and SHP-77 cells, which have very low GJIC although both SRC-418 and STAT3-705 expression levels are low (Figure 2). STAT3 inhibition with S3I-201 in these cells did not reinstate junctional permeability (Table I). Similar results were obtained with CPA7, pointing to a mechanism of gap junction closure which may be independent of the SRC/STAT3 axis.

The fact that cell density up-regulates STAT3 concomitant with the appearance of GJIC prompted us to explore a potential positive role of STAT3 upon GJIC, by assessing the effect of STAT3 inhibition upon GJIC levels in established rat F111 fibroblasts which have extensive communication. As shown in Figure 3A, STAT3 down-regulation through S3I-201 treatment essentially abolished GJIC. Similar results were obtained with CPA7 (not shown). Therefore, rather than increasing GJIC, STAT3 inhibition eliminates gap junctional permeability, that is, STAT3 activity is actually required for gap junction function in immortalised cells which display extensive GJIC.

View this table:
Table I.

Expression of SRC, STAT3 and GJIC in lung cancer lines.

Discussion

We previously demonstrated that cell confluence causes a dramatic increase in STAT3, tyr705 phosphorylation and activity (30). Since SRC is a potent STAT3 activator, we examined its effect upon confluence-mediated STAT3 activation. Interestingly, our results revealed that cell density does not affect SRC-418 levels, while SRC inhibition experiments indicated that cell density-mediated STAT3-705 increase is independent of SRC action. These findings are consistent with previous data regarding fibroblasts from knockout mice where cSRC, and also the related FYN and YES genes had been genetically ablated. In these cells, increasing cell density caused the same dramatic increase in STAT3-705 as in wild-type cells (17), indicating that cell density-dependent STAT3 activation can occur in the absence of these SRC family kinases. Therefore, at any given time, STAT3-705 levels are the sum of the activation due to cell density alone (non-transformed E10, or SK-LuCi6, QUDB), or cell density plus STAT3 activation triggered by SRC and/or other kinases (SK-LuCi6-Src, E10-Src, Calu6, SW-1573, WT-E, BEN, H1299, Figure 4). Activation of STAT3 through direct cadherin engagement was found to occur despite the presence of SRC, which is known to induce cadherin degradation (33); apparently, the residual cadherin present is still able to activate STAT3 above and beyond the activation by SRC (unpublished data).

Since the activity of STAT3 increases dramatically with cell density, the fact that SRC activity is unaffected by cell density implies that although SRC is a known potent STAT3 activator which is involved in cell adhesion signalling, other kinase(s) must be responsible for the cell density-induced increase in STAT3-tyr705 phosphorylation (8, 17, 21). In fact, previous data demonstrated that engagement of the E-cadherin, cell-to-cell adhesion molecule in HC11 mouse breast epithelial cells (30), and of cadherin-11 in Balb/c3T3 fibroblasts (34), causes a dramatic increase in the levels of RAC GTPase. This leads to an increase in secretion of IL6 family cytokines, potent STAT3 activators (30, 35). In fact, Rac1 down-regulation in SK-LuCi6 cells led to a significant reduction in STAT3-tyr705 phosphorylation (Figure 5), pointing to the possibility that the RAC/IL6 pathway may be responsible for the cell density-induced STAT3 up-regulation in these cells as well. In any event, these data are consistent with previous findings (7) demonstrating that SRC inhibition did not reduce STAT3 activity in mouse xenografts of NSCLC cells and point to the importance of STAT3 itself as a therapeutic target.

A large number of membrane tyrosine kinase oncogenes are known to reduce GJIC and could be involved in gap junction closure in NSCLC lines with low SRC activity, by phosphorylating gap junction proteins, directly or indirectly. Similarly to SRC, these oncogenes might activate STAT3. Although STAT3 apparently promotes junctional permeability in normal cells (12, 13), SRC-mediated, STAT3 activation does not lead to an increase in GJIC. This could be due to CX43 phosphorylation by SRC or its effectors or other tyrosine-kinase oncogenes (1, 36) such that the net effect of oncogene action is gap junction closure.

View this table:
Table II.

Effect of Dasatinib treatment upon SRC-418 and STAT-705, at different cell densities

A number of studies demonstrated that STAT3 activates a number of anti-apoptotic genes (5). Induction of apoptosis was shown to lead to a loss of cell coupling, probably due to caspase-3-mediated degradation of CX43 (37). In fact, STAT3 inhibition in cells transformed by SRC (24) or the large tumor antigen of Simian virus 40 (21) leads to apoptosis (38), possibly due to activation of the transcription factor E2F family (potent apoptosis inducers) by these oncogenes. Therefore, apoptosis induced through STAT3 down-regulation in cells with high SRC/E2F activity may accentuate gap junction closure. In lines such as LC-T which have low SRC-418 expression, other oncogenes that activate STAT3 may also activate the E2F family and induce apoptosis, with gap junction closure as a result. Since STAT3 was also previously shown to bind to and activate the CX43 promoter (39), it is also possible that STAT3 inhibition may reduce CX43 mRNA levels directly. In any event, our results demonstrate that although STAT3 is generally growth-promoting, it does not transmit gap junction-repressing signals. This holds true for cells where SRC may have been responsible, at least in part, for GJIC suppression as in the majority of NSCLC lines, but also for lines like LC-T, where SRC levels were found to be low; that is, other oncogenes must be responsible for gap junction closure. On the contrary, STAT3 is required for GJIC in non-neoplastic cells. In cells such as BH-E and SHP77, gap junction closure may be independent of the SRC/STAT3 axis altogether.

Acknowledgements

We would like to thank Dr. Mike Baird and Shallyn Littlefield for CPA7 synthesis, Dr Xiaolong Yang of Queen's University for a gift of the H1299 cells, Dr. Barbara Campling of Queen's University for a gift of cell lines, Dr. James Turkson of the University of Hawaii for gifts of S3I-201, PD180970 and PP2 inhibitors, Dr. David Kaplan of the University of Toronto for the Adenovirus vectors and Kevin Firth, P.Eng., of Ask Sciences products, Kingston, Ontario for engineering design. The financial assistance of the Canadian Institutes of Health Research, the Canadian Breast Cancer Foundation (Ontario Chapter), the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Breast Cancer Research Alliance, the Ontario Centers of Excellence, the Breast Cancer Action Kingston and the Clare Nelson bequest fund through grants to LR is gratefully acknowledged. SG was supported by an NSERC studentship and a Queen's University Graduate Award. MG was supported by a postdoctoral fellowship from the US Army Breast Cancer Program, the Ministry of Research and Innovation of the Province of Ontario and the Advisory Research Committee of Queen's University.

  • Received July 25, 2013.
  • Revision received September 19, 2013.
  • Accepted September 20, 2013.

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Effects of SRC and STAT3 upon Gap Junctional, Intercellular Communication in Lung Cancer Lines
MULU GELETU, STEPHANIE GUY, LEDA RAPTIS
Anticancer Research Oct 2013, 33 (10) 4401-4410;
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