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DU-145 prostate carcinoma cells that selectively transmigrate narrow obstacles express elevated levels of Cx43

The formation of aqueous intercellular channels mediating gap junctional intercellular coupling (GJIC) is a canonical function of connexins (Cx). In contrast, mechanisms of GJIC-independent involvement of connexins in cancer formation and metastasis remain a matter of debate. Because of the role of Cx43 in the determination of carcinoma cell invasive potential, we addressed the problem of the possible Cx43 involvement in early prostate cancer invasion. For this purpose, we analysed Cx43-positive DU-145 cell subsets established from the progenies of the cells most readily transmigrating microporous membranes. These progenies displayed motile activity similar to the control DU-145 cells but were characterized by elevated Cx43 expression levels and GJIC intensity. Thus, apparent links exist between Cx43 expression and transmigration potential of DU-145 cells. Moreover, Cx43 expression profiles in the analysed DU-145 subsets were not affected by intercellular contacts and chemical inhibition of GJIC during the transmigration. Our observations indicate that neither cell motility nor GJIC determines the transmigration efficiency of DU-145 cells. However, we postulate that selective transmigration of prostate cancer cells expressing elevated levels of Cx43 expression may be crucial for the “leading front” formation during cancer invasion.


INTRODUCTION
Connexins (Cx) represent a family of membrane proteins that spontaneously form hexamers, called connexons. The primary function of connexons is the formation of aqueous channels between the adjacent cells, which mediate direct intercellular transfer of small (< 1.5 kDa) metabolites (gap junctional intercellular coupling; GJIC [1]). However, connexins and connexons affect a range of cellular processes in a manner independent of GJIC. For instance, Cx43 and unpaired Cx43 connexons were demonstrated to inhibit cell proliferation [2][3][4][5] and to enhance directed cell motility [6,7] in a manner potentially dependent on their ability to interact with cytoskeletal components [8][9][10]. GJIC plays a crucial role in tissue homeostasis and its impairment in connexindeficient cell populations is involved in the formation of neoplastic loci and primary tumours [11,12]. On the other hand, connexins can enhance the invasive potential of cancer cells in a GJIC-dependent as well as GJICindependent manner [13][14][15]. While GJIC between cancer and endothelial cells was shown to facilitate cancer cell penetration of the endothelial layer [16,17], a GJIC-independent function of Cx43 in brain tumour invasion was reported [14]. These observations suggest the stage-specific function of connexins in the process of cancer development [15]. Impaired connexin expression and GJIC may facilitate early cancer development. However, the re-expression of connexins occurring within specific subsets of heterogeneous cancer cell populations results in the aberrant exchange of intercellular signals between normal and tumour cells, facilitating cancer invasion. In the light of recent data on coupling-independent functions of connexins [4,18], the question arises about the role of connexins in the formation of cancer cell subpopulations constituting a "leading-front" of early invasion. Heterogeneous cell populations [19,20] constituting primary solid tumours comprise subsets of motile cells characterized by the capability of invading surrounding tissues and of metabolic coupling with normal cells. Their early selection seems crucial for subsequent formation of metastases. When recapitulated in vitro, such a selection enables identification of basic cellular properties crucial for early cancer progression [21]. In the present study, we describe an experimental approach enabling the assessment of heterogeneity of prostate carcinoma cells with regard to Cx43 expression and their invasive potential. We demonstrate that the progeny of heterogeneous DU-145 cells most readily transmigrating narrow obstacles is enriched in cells expressing elevated levels of Cx43 and increased GJIC competence. These data indicate that the selection of metastatic cancer cells capable of metabolic coupling with normal tissue cells may occur already during early cancer invasion.

DU-145 cell culture and transmigration experiments
Human prostate carcinoma DU-145 cells were cultivated in DMEM-F12 HAM medium supplemented with 10% FBS (all from Sigma). When indicated, medium containing of 18-α-glycyrrhetinic acid (AGA; Sigma) -a blocker of GJIC -was added to the medium upon cell seeding. In transmigration experiments, DU-145 cells were seeded into chambers containing microporous membranes (Corning; pore diameter 8 μm; membrane diameter 6.5 mm) at the density of 300 cells/mm 2 in the medium alone or the medium supplemented with AGA and allowed to transmigrate for the next 48 hrs, i.e. the time sufficient for the first cells to precipitate onto the well bottom. Their progeny (DU-145_48) was propagated and used for endpoint experiments, along with the progeny of the cells that transmigrated between the 48 th and 72 nd hour of the experiment and precipitated onto the bottom of another well (DU-145_72 cells; see Fig. 2).
siRNA inhibition of Cx43 expression DU-145 cells were seeded at a density of 7x10 4 cells per well in a 12-well plate in antibiotic-free DMEM-F12 HAM medium supplemented with 10% FBS and grown overnight at 37 o C before transfection. The cells were transfected with MISSION ® esiRNA (114 pmol, Sigma) using Lipofectamine TM 2000 (Invitrogen) according to the manufacturer's protocol for 24 hrs. Afterwards, the medium was replaced by standard DMEM-F12 HAM supplemented with 10% FBS and gentamicin, and the efficiency of the inhibition of Cx43 expression was subsequently analysed using immunoblotting along with the estimation of the motility of transfected cells with time-lapse microscopy.

Time-lapse monitoring of movement of individual cells
The movement of DU-145 cells was time-lapse recorded for 4 hrs and the tracks of individual cells were determined from the series of changes in the cell centroid positions, pooled and analysed as previously described [22,23]. The following parameters were estimated: (i) the total length of cell displacement (µm), i.e. the distance from the starting point directly to the cell's final position, (ii) the rate of cell displacement (µm/h), i.e. the distance from the starting point directly to the cell's final position/time of recording (4 hours), (iii) the speed of cell movement (μm/h), i.e. total length of cell trajectory/time of recording (4 hrs). Cell trajectories from no less than three independent experiments (number of cells > 50) were taken for the estimation of statistical significance by the non-parametric Mann-Whitney test # p < 0.01.

Immunocytochemistry, immunoblotting and GJIC analyses
Actin cytoskeleton architecture was analysed in formaldehyde-fixed, Triton X-100-solubilised DU-145 cells stained with TRITC-conjugated phalloidin (Sigma). For immunocytochemical analyses of intracellular localization of Cx43, cells were fixed with methanol:acetone (7:3, -20 o C), labelled with rabbit anti-Cx43 IgG (Sigma) and counterstained with Alexa 488-conjugated goat antirabbit IgG (Invitrogen, Carlsbad, CA) and 0.5 µg/ml bis-benzimide (Hoechst) [24]. Visualization of Cx43-positive plaques and semi-quantitative evaluation of their abundance at intercellular contacts was performed with a Leica DMIRE2 microscope. For Western blot analyses, DU-145 cell cultures were dissolved in lysis buffer and cellular proteins (30 μg/lane) were applied to 15% SDSpolyacrylamide gels, followed by transfer to nitrocellulose. Blots were exposed to primary rabbit polyclonal anti-Cx43 and mouse monoclonal anti-α-tubulin antibody (both from Sigma) followed by detection of the antibodies using HRPlabelled secondary antibodies (Invitrogen) and SuperSignal West Pico Substrate (Pierce, Rockford, IL) [25]. GJIC intensity was measured as previously described [26] with some modifications. Briefly, donor cells labelled with calcein (Molecular Probes) were plated onto monolayers of acceptor cells grown in Petri dishes at a ratio of l:50. The dynamics of calcein transfer from donor to acceptor cells was visualised using a Leica DM IRE2 time-lapse system and analysed with ImageJ freeware. For this purpose, values of the averaged coupling ratio (c r ), i.e. the number of calcein-positive recipient cells per donor cell, were elucidated 24, 30, 36 and 42 min. after donor cell seeding. At least 50 donor cells per Petri dish analysed in three independent experiments performed for each cell population were taken for the estimation of statistical significance by Student's t-test; * = p < 0.05.

Correlation between Cx43 plasmalemmal localization and transmigration activity of DU-145 cells
Immunofluorescence studies revealed heterogeneous expression levels and patterns of Cx43 localization in single DU-145 cells. Cx43 could be observed in perinuclear regions of their cytoplasm, in the cell protrusions and in plaques localized at intercellular contacts (Fig. 1A). Similarly, DU-145 cells displayed heterogeneous motile activity. This is shown in the circular diagram of cell trajectories (Fig. 1B) and the frequency histogram of cell displacements in sparse cultures (Fig. 1C), where the fractions of motile and immobile cells could be discriminated. Thus, the DU-145 cell line comprises sub-populations differing in cell motility and expression pattern of Cx43, two features involved in determination of the invasive potential of cancer cells. Transmigration tests were further performed to establish the invasive subsets of DU-145 cells and to elucidate the correlation of Cx43 expression on their invasive potential. The DU-145_48 subset representing the progeny of the cells most readily transmigrating microporous membranes (2.24% of initial cell population; Fig. 2B) displayed elevated numbers of Cx43-positive plaques at cell-to-cell contacts (  The progenies of cells that managed to precipitate onto the bottoms of the wells were further analysed using fluorescence and time-lapse microscopy (see Fig. 4). DU-145_48 and DU-145_72 populations (E and F, respectively) were characterised by considerably higher abundance of Cx43-positive plaques than control cell populations (D, semiquantitatively summarised in Tab. 1). Bar -25 μm. Tab. 1) propagated from the progeny of the "second wave" of transmigrating DU-145 cells (2.42% of initial cell population; Fig. 2C). On the other hand, immunoblot analyses demonstrated increased Cx43 expression levels in DU-145_48 but not in DU-145_72 cell populations (Fig. 3A, left panel). These data indicate that the subpopulations of DU-145 cells characterized by elevated levels of plasmalemmal Cx43 preferentially transmigrate narrow obstacles. Since transmigration experiments were performed in conditions partly securing physical isolation of the cells (300 cells/mm 2 ), it can be stated that the transmigration efficiency of DU-145 cells was not affected by the functional status of gap junctional channels. To substantiate this notion, transmigration tests were performed in the presence of 18-α-glycyrrhetinic acid (AGA). When    in the regions adjacent to cellular protrusions (Fig. 4G-I). These data demonstrate lack of a correlation between Cx43 expression and the motility of DU-145 cell subsets. This notion was further substantiated by siRNA experiments which demonstrated that transient inhibition of Cx43 expression (Fig. 5A) exerted only a minute effect on control DU-145 cell motility. The motility of DU-145 cells analysed 72 hours after transfection, i.e. when the pronounced inhibition of Cx43 expression was observed, was similar to the motility of control cells regardless of their density (Fig. 5B-E). This observation indicates a lack of functional link between Cx43 expression and heterogeneous locomotion of DU-145 cells during the process of transmigration. Thus, neither motile activity of DU-145 cells nor differences in the architecture of their cytoskeleton can account for the correlation between the abundance of plasmalemmal fraction of Cx43 and DU-145 cell ability to penetrate narrow obstacles.
On the other hand, elevated numbers of Cx43-positive plaques at intercellular contacts between invasive DU-145 cells correlated with increased efficiency of GJIC in DU-145_48 and DU-145_72 cell populations ( Fig. 6B and C, respectively; cf. A). This is illustrated by a more than three-fold increase of the average c r in DU-145_48 cell populations compared to control DU-145 cells 42 min. after donor cell seeding (Fig. 6D). These data indicate that transmigration of DU-145 cells results in the formation of well-coupled DU-145 subsets.

DISCUSSION
During cancer progression, subpopulations of "invasive" cells are formed within an expanding and phenotypically heterogeneous tumour cell population [27]. Due to their specific properties, such as high mechanical elasticity, active motility and the capability of tissue remodelling and of communication with local microenvironments [28][29][30], these cells are apt to effectively cross tissue barriers [31,32] and to form a "leading front" of cancer invasion. Heterogeneity of cancer cells observed in vivo was also found within in vitro cultured cancer cell populations [13,[32][33][34]. We demonstrate such heterogeneity of DU-145 cell populations with regard to Cx43 expression. More importantly, we show that the capability of DU-145 cells to transmigrate narrow obstacles coincides with high expression levels of Cx43 in these cells. Thus, selective cell transmigration in the conditions mimicking early cancer invasion results in the formation of cellular subsets readily forming functional gap junctions. Due to the crucial role of metabolic coupling between cancer and normal cells in cancer progression [17,35], it may increase the efficiency of cancer cell dissemination from primary tumours. Transmigration tests were performed in conditions securing the physical isolation of the cells. Moreover, pharmacological block of gap junctional coupling exerted no effect on the Cx43 expression profile in the progeny of transmigrating cells. Thus, a fraction of DU-145 cells can preferentially transmigrate narrow obstacles in the presence of non-functional connexons. The question arises about the possible ways of Cx43 participation in the determination of DU-145 cell transmigration potential. A GJIC-independent function of Cx43 in the regulation of cell locomotion has been previously described [6,14]. However, we did not observe any correlation between the expression and function of Cx43 and motility of DU-145 cells. The progeny of transmigrating cells, while expressing elevated levels of Cx43, displayed motility similar to control cells. This observation indicates that if Cx43 is directly involved in the determination of DU-145 cell transmigration potential, it exerts this effect in a manner independent of cell motility. In parallel, it remains in opposition to previous reports describing effects of Cx43 on cell adhesion and directed motility [3,4,6,10,14,18]. However, the processes of cell migration on planar surfaces and cell transmigration through narrow obstacles may be governed by discrete regulatory systems [30,36]. It is possible that subtle changes in cytoskeletal function that facilitate cellular susceptibility to mechanical distortions [29,37] and/or directed cell motility through narrow obstacles may accompany elevated Cx43 expression. This explanation is consistent with the reports on the interactions of connexins with cytoskeleton proteins [4,10], resulting in a protective effect against mechanical injury of cancer cells [8]. Importantly, it would explain the formation of cancer cell subpopulations characterised by heterogeneous motile activity on planar substrata and displaying high competence for GJIC -a parameter crucial for penetration of tissue barriers such as endothelium [15-17, 35, 38]. While direct involvement of Cx43 in the transmigration of DU-145 cells remains a matter of speculation, the coincidence between plasmalemmal localisation of Cx43 and transmigration potential of DU-145 cells may have strong implications for our understanding of its involvement in cancer progression. Cancer cells have developed a range of strategies facilitating their penetration of the connective tissue and the process of intra-and extravasation. They remain under the control of versatile regulatory systems including cell adhesion and shape, function of the cytoskeleton [30] and rearrangements of the cell "niche" [39,40] resulting in the formation of migratory trails [30,41]. Our observations support the notions about Cx43 as an important element of this "puzzle". They indicate that the selective migration of cancer cell subpopulations competent for GJIC with normal cells and thus more apt to invade healthy tissues may occur already at early stages of cancer invasion.