Rac1-mediated cytoskeleton rearrangements induced by intersectin-1s deficiency promotes lung cancer cell proliferation, migration and metastasis

A549 cells were grown in HAM’s F12 medium, containing 10 % FBS, 1 % 1-Alanyl-L-glutamine, and 1 % antimycotic, at 37 °C in a 5 % CO₂ incubator. Bronchial cells were grown in airway epithelial basal cell medium (0.25 % HLL, 3 % L-glutamine, 0.4 % Extract P, 1 % airway epithelial cell supplement). Subconfluent A549 cells were transfected with a myc-tagged full-length ITSN-1s DNA cloned into the mammalian vector pcDNA3.1Myc/His (GeneCopoeia™, Rockville, MD), using 7:2 ratio of Fugene HD transfection reagent (μl) to DNA (μg) [26]. For stable transfection, ITSN-1s transiently transfected cells were grown in 100 cm² dishes with 1 mg/ml G418. HAM-F’12 medium containing 1 mg/ml G418 was replenished every other day for about 2 weeks until stable transfected A549 cells began to grow. At that point the concentration of G418 in HAM’s F12 medium was switched to 800 μg/ml for a week and then maintained at 500 μg/ml. For siRNA transfection, a set of 4 siRNA targeting Cbl gene, control siRNA and Transfection Reagent from Dharmacon (Lafayette, Co) were used; 0.1×10⁶ cells in 2 ml complete medium without antibiotics were seeded in 6-well plates and grown to 70 % confluence prior to transfection. Control siRNA and Cbl siRNA (5 μM) were diluted in transfection medium (4 μl) as per manufacturer’s instructions and added dropwise over the cells. Transfected cells were incubated at 37 °C in a 5 % CO₂ for 8 h without changing medium. Then the medium was replaced with normal growth medium. Optimal siRNA/transfection reagent ratio and transfection time were determined in pilot studies.

Specific Antibodies (Ab) were as follows: ITSN-1, Eps8 (BD Biosciences, San Jose, CA); mSos1, Rac1, Cbl, control IgG, Ub, vinculin, vimentin (Santa Cruz Biotechnology, Santa Cruz, CA); ITSN-1, Actin (Sigma-Aldrich, St. Louis, MO). All fluorophor-conjugated Abs and the Prolong Antifade reagent were from Molecular Probes (Eugene, OR).

Lung tissues and cells were lysed using the Qiagen QIA Shredder homogenizer; total RNA was isolated using the RNeasy Mini RNA isolation kit (Qiagen, Valencia, CA). The cDNA was synthesized from 2 μg RNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). RT-PCR was performed on an Applied Biosystems 7900HT machine [17]. The relative mRNA levels were normalized to housekeeping gene cyclophillin, and determined by calculating the ΔΔCt value as per manufacturer’s guidelines.

Anchorage-independent growth was determined by assaying colony formation in soft agar [28]. Briefly, 1 × 10⁵ A549 and A549 + ITSN-1s cells resuspended in 5 ml HAM’s F12 medium (containing 10 % FBS and 0.28 % Difco Nobal Agar) were plated in triplicates onto 35 mm dishes over a 2.5 ml layer of solidified HAM’s F12/10 % FBS and 0.58 % agarose. The cells were incubated in humidified 5 % CO₂, 95 % air, at 37 °C and fed by adding 2 ml of HAM’s F12/10 % FBS every 2–3 days. Thirty days after seeding, colonies were stained with 0.01 % crystal violet dye and counted. Pictures were taken by Alpha Imager HP digital scanner.

A549 cells and A549 + ITSN-1s cells were subjected to MTT cell proliferation [17]. Briefly, triplicate aliquots of cells (10⁶ cells suspended in 100 μl complete medium) were serially diluted in medium, onto a 96-well plate. After 48 h, 10 μl MTT Reagent were added to each well. After 5 h incubation, 100 μl of detergent was added, the plate was covered and kept in the dark at room temperature (RT) overnight. The next day the absorbance at 570 nm was determined using a Dynex microplate reader. Parallel triplicate experiments using non-treated cells were performed; cells were counted using a hemocytometer and a growth curve was generated to relate the OD⁵⁷⁰ values to the cell number per well.

Cells were lysed in a buffer containing 50 mM Tris-HCl-pH8.0, 150 mM NaCl, 1 % NP-40, 1 mM Na₃VO₄, 1 mM PMSF, and protease and phosphatase inhibitors for 2 h at 4 °C. Lysates were centrifuged (45 min, at 45000 rpm, at 4 °C) and stored at −20 °C. Protein content was determined using Micro Bicinchoninic acid assay. For immunoprecipitation (IP), cell lysates (500 μg of total protein) were pre-cleared using 25 μl protein A/G slurry for 30 min at RT. The supernatants were then incubated with 3 μg mSos1, Cbl or isotype matched IgG Abs for 1 h at RT. 40 μl of protein A/G slurry (Santa Cruz Biotechnology, Santa Cruz, CA) were then added to the supernatants and incubated at 4 °C overnight. The bound proteins were resolved on 5–20 % SDS-PAGE. Western blot (WB) was performed using primary Abs and appropriate secondary Abs [26]. TrueBlot® ULTRA secondary Ab (Rockland Immunochemicals, Limerick, PA) was used for Rac1 to avoid background from the light chain band. For detection of Eps8 ubiquitination by WB, 5 mM N-ethylmaleimide was added in the IP buffer to prevent the cleavage of polyubiquitin chains [9].

A549 cells and A549 + ITSN-1s cells were grown under similar conditions, trypsinized and counted. Equal number of cells were lysed and centrifuged for 5 min at 10,000xg. The pellet (vimentin full length filaments) and 60 μg total protein of supernatant (collapsed vimentin filaments) were solubilized in equal volume of Laemmli sample buffer, denatured by boiling, and equal volumes were resolved on 5–20 % SDS-PAGE and analyzed by WB with vimentin Ab [29].

Immunofluorescent and phalloidin staining of cells grown on coverslips was performed [30]. Briefly, cells were seeded on non-collagen coated coverslips in 6-well plates. Cells were washed three times with cold PBS, followed by fixation and permeabilization with methanol at −20 °C for 5 min. Methanol was immediately removed and blocking buffer (PBS with 1 % BSA) was added for 45 min at RT. Following removal of blocking buffer, cells were incubated with primary Ab (ITSN-1 Ab (1:250), vinculin Ab (1:250) or vimentin Ab (1:250) in 0.1 % BSA/PBS) for 1 h at RT. The cells were washed with 0.1 % BSA in PBS three times for 10 min each. Coverslips were then incubated with appropriate secondary Ab (at dilution 1:500 in 0.1 % BSA/PBS) for 1 h, protected from light, at RT followed by washing as above. For phalloidin Alexa Flour (AF) 488 staining, cells were fixed in 3.7 % paraformaldehyde in PBS for 15 min at RT, permeabilized in 0.1 % Triton X-100 for 5 min on ice, and stained with phalloidin AF488 for 30 min at RT. Cells were mounted with Prolong Antifade reagent. For morphometry, the number of vinculin clusters per 250 μm² was counted in 3 randomly chosen areas along the cell perimeter and was averaged from different experiments performed in triplicates. At least 50 cells were counted per experiment. Micrographs were taken with a Zeiss AxioImager M1 microscope equipped with a digital camera. All images used for quantification were acquired using identical parameters per experiment.

Immunohistochemistry (IHC) on paraffin-embedded LC specimens and TMA using the ITSN-1 Ab was performed as previously described by us [17]. ITSN-1s Ab from Sigma-Aldrich (St. Louis, MO) was used for immunofluorescence and IHC.

Briefly, cells were washed in PBS and collected into 1.5 ml Eppendorf tubes. Cell pellets were then resuspended in 100 μl of lysis buffer I (20 mM Hepes-NaOH, pH 7.2, 100 mM NaCl, 1 mM sodium orthovanadate, 50 mM NaF, 1 % Triton X-100, protease inhibitors) for 1 h and centrifuged for 20 min at 22,000 rpm [30]. Supernatants were saved as G-actin fractions; the pellet was resuspended in lysis buffer II (15 mM Hepes-NaOH, pH 7.5, 0.15 mM NaCl, 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % SDS, 10 mM EDTA, 1 mM dithiothreitol, 1 mM sodium orthovanadate, protease inhibitors) for 1 h and then centrifuged at 45,000 rpm for 40 min. The ensuing supernatants represented the F-actin fractions. Equal protein amounts were loaded onto 4–12 % SDS-PAGE. Actin was detected by immunoblotting with actin Ab and quantified by densitometry.

Cells were seeded into 12-well plates and grown to 90–100 % confluence. A sterilized micro-pipette tip was used to generate a scratch across the cell layer. Cells at multiple points along the scratch were imaged every 15 min over a 48 h period using time-lapse microscopy using a Zeiss AxioVert Z1 microscope. Images were captured at 100× magnification. Cell migration measurements were made using NIH imageJ software.

Protocols were approved by the Institutional Animal Care and Use Committee and Institutional Biosafety Committee of Rush University. Five week old immunodeficient male mice (Jackson Labs, ME) were used. For metastasis assay, mice were injected retro-orbitally with 2 × 10 ⁶ cells in 0.1 ml PBS. After 12 weeks mice were sacrificed. The lungs were removed, fixed in 4 % formaldehyde/PBS, paraffin-embedded, dissected and stained with hematoxylin and eosin (H&E) (27). For the xenograft tumor assay, 10⁷ cells resuspended in PBS were mixed with matrigel (BD Bioscience, Bedford, MA) at a 1:1 volume ratio in 200 μl of medium without serum and injected subcutaneously into the flanks of mice [31]. After 4 weeks, mice were sacrificed and the tumor was resected. Pictures were taken and the tumor area was measured NIH imageJ software.

Cell lysates prepared in ice-cold lysis buffer were centrifuged (10,000 g, 4 °C for 1 min) to remove cell debris and then incubated with 40 μl of p21 activated kinase (PAK)-GST beads [Cdc42/Rac1 activation kit (Cytoskeleton, Denver, CO)] or 50 ml of Rhotekin-GST beads for RhoA for 60 min at 4 °C, as per manufacturer’s instructions. Beads were washed and boiled for 2 min in 20 μl of Laemmli sample buffer; samples were run in parallel with total cell lysates and GTP/GDP control, and WB performed with Cdc42/Rac1/RhoA Ab. Activation of Cdc42/Rac1/RhoA in control and transfected cells was determined by densitometry.

All findings were confirmed in three to five different experiments performed under identical conditions. Images were acquired using identical parameters. Densitometry analysis was performed using NIH ImageJ software. Data are expressed as mean or median ± SE. Comparisons were made using unpaired student’s t-test. P values less than 0.05 were considered statistically significant.

To address whether ITSN plays a role in LC, we examined ITSN-1s protein level in human LC cells by WB with ITSN-1 Ab compared to human bronchial cells (Fig. 1a). Downregulation of ITSN-1s protein level was consistent for all LC cell lines (Fig. 1a, lanes b – f vs. a). Densitometry indicated that the extent of downregulation ranged from 42 % to undetectable levels in H1437 adenocarcinoma cells (Fig. 1a, e). To determine if downregulation of ITSN-1s is due to inhibition of transcription or post-translational modifications, qPCR analyses were performed. ITSN-1s mRNA levels were assessed in A549 cells compared to bronchial cells, and in adenocarcinoma tissue (Table 1), compared to non-LC tissue (Fig. 1b). Similar to protein level, ITSN-1s mRNA level was decreased in LC by 38 to 81 %.

To further confirm this finding we examined the level of ITSN-1s in 10 lung adenocarcinoma tissue samples along with adjacent normal tissue. IHC analyses revealed high ITSN-1s immunostaining in the normal bronchial epithelial cells and adjacent normal alveoli (Fig. 1c, a, a1) but much weaker or even undetectable levels in some tumors (Fig. 1c, b, b1). The level of ITSN-1s was reduced by more than 50 % compared to normal areas in 8 of the adenocarcinoma samples examined. In addition, we evaluated a TMA slide comprising 20 lung adenocarcinoma samples: 10 grade 1 (well differentiated) and 10 grades 2 or 3 (moderate to poorly differentiated) and 20 normal tissue samples. Each TMA spot was examined and graded as having high (Fig. 1d, a) or low ITSN-1s (Fig. 1d, b) protein level. The presence of at least 25 % of cancer cells staining positive (intensity of a minimum of 2 on a scale of 1 to 3 [32, 33]) within a patient sample was considered high ITSN-1s protein level. A high ITSN-1s protein level was noted in 85 % of normal tissue, 30 % of grade 1 LC tissue and none of the grades 2 or 3 LC tissue (Fig. 1e). Altogether these findings provide strong evidence that ITSN-1s protein level is downregulated in LC.

Next, we examined the subcellular distribution of ITSN-1s in A549 cells compared to bronchial cells. Immunofluorescent staining using ITSN-1 AF 488-conjugated reporter Abs showed a wide subcellular distribution of ITSN-1s in bronchial cells; the strong punctate pattern at the plasma membrane and in the cytoplasm as well as in the perinuclear area is consistent with ITSN-1s’ association with the endocytic and Golgi vesicles (Fig. 2a, b, b1), as previously reported in other cell types [26, 34]. A549 cells revealed a similar subcellular distribution of ITSN-1s (Fig. 2a, a, a1), but a decreased staining intensity, consistent with downregulation of ITSN-1s protein level in LC.

To determine the impact of ITSN-1s downregulation on LC, we first restored ITSN-1s protein level in A549 cells using myc-ITSN-1s. To improve and to preserve the level of ITSN-1s long term, we created stable transfected myc-ITSN-1s A549 cells (A549 + ITSN-1s; Fig. 2b). Western blot using myc Ab (Fig. 2b, a) and ITSN-1s Ab (Fig. 2b, b) demonstrated successful transfection. Densitometry indicated restoration of ITSN-1s protein level to 91 % of normal bronchial cells (Fig. 2c). Immunoflorescence staining (ITSN-1s/AF 488 Ab) of A549 + ITSN-1s cells (Fig. 2d, d, d1) compared to A549 cells (Fig. 2a, a, a1), demonstrated a similar subcellular distribution but increased staining intensity, consistent with restoration of ITSN-1s protein level.

Prior studies have shown that ITSN-1s regulates cell proliferation via interaction with Ras [35]. Therefore, we decided to investigate the effects of restoring ITSN-1s protein level on LC cell proliferation. Briefly, A549 and A549 + ITSN-1s cells were seeded in 100 cm² plates in triplicate, cultured for five days and counted. A549 + ITSN-1s cells showed 30 % inhibition of cell proliferation compared to A549 cells (Fig. 3a). A549 and A549 + ITSN-1s cells proliferation was also evaluated by the MTT assay. A growth curve was generated to relate the OD⁵⁷⁰ values to the cell number per well. The OD⁵⁷⁰ values for A549 + ITSN-1s cells were significantly lower compared to A549 cells in all wells of the cell culture plate (Fig. 3b). Three points on the linear part of the proliferation curve were used for quantification of the extent of cell growth inhibition. The results indicated a 20 % inhibition in proliferation of A549 + ITSN-1s cells compared to A549 cells (Fig. 3c).

Since restoring ITSN-1s level impairs A549 cell proliferation, we examined whether ITSN-1s regulates anchorage-independent growth, one of the hallmarks of cell transformation [28]. We performed the soft-agar assay, a validated in vitro assay for detecting malignant transformation [28]. Cells started to form noticeable colonies after 30 days of culture. Although both A549 and A549 + ITSN-1s cells formed colonies, the ability of A549 + ITSN-1s cells to grow independent of anchorage was impaired compared to A549 cells (Fig. 3d); the number of colonies was 80 % lower (Fig. 3e), suggesting that ITSN-1s deficiency is an important component in the anchorage-independent growth of human LC cells.

To gain insight into ITSN-1s’ role in anchorage-independent growth of LC cells in vivo, we performed a xenograft tumor assay [31]. Immunodeficient mice were injected subcutaneously with A549 and A549 + ITSN-1s cells. Tumor development and growth were monitored for 4 weeks at which point tumors were resected, photographed (Fig. 3f), and measured. The tumors of mice injected with A549 + ITSN-1s cells were 42 % smaller than the tumors of mice injected with A549 cells (Fig. 3g). Together these studies demonstrate that ITSN-1s restoration in A549 cells significantly imapirs tumor proliferation and anchorage-independent growth.

To address whether ITSN-1s deficiency interferes with migration of LC cells, we performed a scratch assay which preserves cell-cell interactions and is able to mimic migration of cells in vivo [36], in conjunction with time-lapse microscopy (Fig. 4a). A549 + ITSN-1s cells showed statistically significant inhibition in scratch closure as early as 3 h. The scratch was completely closed by A549 cells at 24 h, whereas, A549 + ITSN-1s cells closed only 60 % of the scratch (Fig. 4b) at this same time point. The scratch closure is due to both cell proliferation and cell migration into the scratch from the periphery. The impact of either proliferation or migration in scratch closure cannot be determined just based on the images, especially given that the cells are grown to confluence prior to creating the scratch and given that cancer cells migrate collectively in sheets/lumps. To determine the impact of increased ITSN-1s protein level on cell migration independent of cell proliferation, cells grown to confluence were pretreated with 7.5 μg/ml of mitomycin C (Sigma-Aldrich, St. Louis, MO) for 1 h which impaired further cell proliferation efficiently without killing the cells (S1, A). Mitomycin C is a widely used antibiotic because of its mild toxicity and potent antitumor activity. Mitomycin C reacts covalently with DNA, forming crosslinks between the complementary strands of DNA. This prevents separation of the complementary strands and thus inhibits DNA replication [37]. Based on the efficiency of inhibition noted with mitomycin C, we believe the additional cells noted in the scratch at 24 h in mitomycin C treated cells (Additional file 1: Figure S1B, 2 panels on the right) is due to predominantly cell migration of remaining cells from the periphery. Mitomycin C treated A549 + ITSN-1s cells compared to A549 controls significantly delayed the scratch closure (S1, B) and overall impaired cell migration by 52 % (Fig. 4c). This finding demonstrates that ITSN-1s plays a significant inhibitory role in cell migration in addition to its antiproliferative effects.

To further validate this finding we used a mouse metastasis assay. A549 and A549 + ITSN-1s cells were injected into the retro-orbital sinus of immunodeficient mice [16]. After 12 weeks, mice were sacrificed and the lungs were prepared for routine morphological analyses [16]. For evaluation of pulmonary metastasis, lungs were cut in 5 μm sections and stained with H&E. Five sections throughout the whole lung per mice were screened histologically, and the number of metastases was counted and classified based on their size as small (diameter <200 μm), medium (diameter 200–500 μm) or large (diameter >500 μm). A549 and A549 + ITSN-1s injected mice developed tumors in the lung parenchyma (Fig. 4d, d1, d5), pulmonary artery (Fig. 4d, d2, d6), sub-pleura (Fig. 4d, d3, d7) and peribronchial lung (Fig. 4d, d4, d8). Mice injected with A549 cells had significantly more tumors per lung section (median 12.5 ± 0.7 vs. 2 ± 0.47, Fig. 4e), as well as more tumors per each size group (median, small 6 ± 0.56 vs. 1 ± 0.12, medium 5 ± 0.47 vs. 1 ± 0.38, large 1 ± 0.39 vs. 0, Fig. 4f) compared to mice injected with A549 + ITSN-1s cells. Together these findings are consistent with an important role of ITSN-1s in restricting LC proliferation, migration and metastasis.

To determine if ITSN-1s was still present in the emergent tumors, we performed immunofluorescence staining of lung sections with ITSN-1s antibody. Tumors of all size in mice injected with A549 cells showed very low to undetectable levels of staining for ITSN-1s (Additional file 2: Figure S2, A, C). However, mice injected with A549 + ITSN-1s cells showed variability in ITSN-1s expression based on the size of the tumors. Smaller tumors showed higher ITSN-1s staining throughout the tumor (Additional file 2: Figure S2, B, b1). In contrast, larger tumors showed more heterogeneous expression with patches of cells showing high ITSN-1s staining and other patches of cells within the same tumor showing low ITSN-1s staining (Additional file 2: Figure S2, D, d1). This supports the role of ITSN-1s as a tumor suppressor.

Since cancer cell migration and metastasis requires reorganization of the cytoskeleton leading to epithelial-mesenchymal transformation [38], we next addressed whether decreased ITSN-1s protein level may be involved in reorganization of the components of the cytoskeleton to favor migration and metastasis. Phalloidin staining revealed small filaments of actin in A549 cells (Fig. 5a), whereas in A549 + ITSN-1s cells it showed thick bundles of actin pointing towards peripheral attachment points (Fig. 5b, b1). In addition, A549 + ITSN-1s compared to A549 cells showed more spreading and loss of elongated and polarized morphology. Cell fractionation and differential centrifugation of A549 and A549 + ITSN-1s cell lysates followed by WB analyses and densitometry showed no change in the G- and F-actin fractions between the two cell lines (Fig. 5c), suggesting that restoring ITSN-1s protein level does not result in formation of new actin or actin polymerization, but simply reorganizes existing actin to form bundles. Since association/dissociation of the actin cytoskeleton structure with FA proteins determines cell motility and metastasis [39], we performed double actin/vinculin immunofluorescent staining (Fig. 5d – i). A549 + ITSN-1s cells had increased actin-vinculin colocalization (Fig. 5f, f1) and a higher number of vinculin clusters per surface area compared to A549 cells (Fig. 5j). The mean number of vinculin clusters per 250 μm² cell surface was 4.24 ± 0.8 vs. 10.4 ± 1.23 in A549 cells and A549 + ITSN-1s cells respectively.

The other component of the cell cytoskeleton involved in metastasis is vimentin intermediate filaments [40]. Immunofluorescence staining and image analyses showed a wide spread vimentin filament network in A549 cells (Fig. 6a). A549 + ITSN-1s cells showed an altered subcellular distribution of vimentin with collapse of vimentin intermediate filaments into clusters of short filaments near the nucleus (Fig. 6b). Next, WB using vimentin Ab was applied on A549 and A549 + ITSN-1s cells lysates as described under Methods. Full-length vimentin filaments which are not soluble in lysis buffer are present in the pellet, whereas the collapsed intermediate and small filaments are soluble and present in the supernatant. Densitometry analyses indicated decreased insoluble vimentin and increased soluble vimentin in A549 + ITSN-1s cells compared to A549 cells (Fig. 6c), consistent with our morphological findings. Based on these findings and previous reports indicating that the collapse of vimentin filaments inhibits cancer progression [41], we concluded that restoring ITSN-1s protein level stabilizes the cells and contributes to decreased motility and metastasis.

Changes in cell cytoskeleton organization are mediated by the GTPase proteins and it has been shown that ITSN-1s interacts with mSos1, a GEF for Rac1, and with CdGAP, a GAP for Cdc42 [6, 22, 23, 35]. We therefore addressed whether ITSN-1s may regulate the activation status of these proteins. A549 and A549 + ITSN-1s cell lysates were subjected to Rac1, Cdc42 and RhoA activation assays (Fig. 7a). Densitometry demonstrated a 32 % decrease in activated Rac1 and 18 % increase in RhoA but no significant difference in activated Cdc42 in A549 + ITSN-1s cells compared to A549 cells (Fig. 7b). These findings are consistent with previous reports indicating a reciprocal balance between Rac1 and RhoA [42, 43].

Rac1 is activated by interacting with mSos1-Eps8 complex which is formed in the presence of activation of RTK [44]. Since ITSN-1s is an mSos1 interacting protein, we performed IP of A549 and A549 + ITSN-1s cell lysates with mSos1 Ab and WB with Rac1 Ab to evaluate if restoring ITSN protein level interferes with mSos1-Rac1 interaction. No detectable difference was seen in the mSos1-Rac1 interaction between A549 and A549 + ITSN-1s cells (Fig. 7c, upper panel). The lower panels in Fig. 7c indicate the protein level of Rac1 and mSos1 in A549 and A549 + ITSN-1s lysates. As Eps8 is the other component of the complex required for Rac1 activation, we next evaluated if the level of ITSN protein level impacted mSos1-Eps8 interaction. IP using mSos1 Ab showed less mSos1-Eps8 interaction in A549 + ITSN-1s cell lysates compared to A549 cells (Fig. 7d, upper panel). Moreover, WB of cell lysates revealed that the level of Eps8 protein is lower in A549 + ITSN-1s compared to A549 lysates, while the level of mSos1 protein is unchanged (Fig. 7d, lower panels). Densitometry indicated that restoring ITSN-1s decreased Eps8 protein level by 20 % and impaired the effective formation of mSos1-Eps8 complex by 54 % (Fig. 7e).

ITSN-1s is known to interact with Cbl and enhance its activity leading to ubiquitination of important signaling proteins [7, 8]. Therefore, we investigated if decreased Eps8 protein level in A549 + ITSN-1s is due to its ubiquitination and degradation. Cells pretreated with 10 μM of MG132 (Sigma-Aldrich, St. Louis, MO) for 2 h and untreated cells were lysed and IP performed with Eps8 Ab, followed by WB with both Eps8 (Fig. 8a, lower panel) and ubiquitin (Fig. 8a, upper panel) Abs. MG132 is a specific and potent proteasome inhibitor which reduces the degradation of ubiquitin-conjugated proteins [45]. Untreated A549 + ITSN-1s cells demonstrated less full-length Eps8 compared to A549 cells and additional bands above (Fig. 8a, arrows), which represent the ubiquitinated Eps8 protein. The same protein bands were immunoreactive to ubiquitin Ab (Fig. 8a, bracket). Pretreatment with MG132 resulted in restoration of Eps8 protein level (Fig. 8a, lower panel) and marked accumulation of ubiquitinated Eps8 (Fig. 8a, upper panel), indicating that ITSN-1s mediated downregulation of Eps8 is via proteasomal degradation. In the presence of MG132, A549 + ITSN-1s cells showed relatively limited accumulation of ubiquitinated Eps8 when compared to A549 cells (Fig. 8b). To confirm that Eps8 ubiquitination is via Cbl, we performed IP with Cbl Ab and WB with Eps8 Ab (Fig. 8c) which demonstrated increased interaction between Cbl and Eps8 in A549+ ITSN-1s cells (Fig. 8c, upper panel). The lower panels in Fig. 8c indicate the level of Cbl and Eps8 in the A549 and A549 + ITSN-1s lysates. Densitometry analyses indicate that ITSN restoration improved the effective formation of Cbl-Eps8 complex by 83 % (Fig. 8d). To further validate that Eps8 degradation is via Cbl, we used a siRNA approach to knockdown Cbl protein level. A549 and A549 + ITSN-1s cells were transfected with Cbl-siRNA and control-siRNA (Fig. 8e). Cbl protein level was unaffected by control-siRNA, whereas Cbl-siRNA efficiently knocked down the Cbl protein level in A549 and A549 + ITSN-1s cells (Fig. 8e, middle panel). Moreover, transfection with control-siRNA showed degradation of Eps8 in A549 + ITSN-1s cells (Fig. 8e, upper panel-left 2 lanes). However, with Cbl knockdown, Eps8 degradation in A549 + ITSN-1s cells was prevented (Fig. 8e, upper panel-right 4 lanes).

In summary, our studies show that restoring ITSN-1s increases Cbl-Eps8 interaction leading to Eps8 ubiquitination and degradation. The preferential formation of Cbl-Eps8 contributes to impaired assembly of Eps8-mSos1 complex, leading to Rac1 inactivation, reorganization of actin into thick bundles, increased FA complexes and collapse of the vimentin filament network altogether leading to decreased LC cell migration and metastasis (Fig. 9).