Epithelial-to-mesenchymal transition promotes tubulin detyrosination and microtentacles that enhance endothelial engagement

Abstract

Epithelial-to-mesenchymal transition (EMT) is associated with increased breast tumor metastasis; however, the specific mechanisms by which EMT promotes metastasis remain somewhat unclear. Despite the importance of cytoskeletal dynamics during both EMT and metastasis, very few current studies examine the cytoskeleton of detached and circulating tumor cells. Specific posttranslational α-tubulin modifications are critical for adherent cell motility and implicated in numerous pathologies, but also remain understudied in detached cells. We report here that EMT induced through ectopic expression of Twist or Snail promotes α-tubulin detyrosination and the formation of tubulin-based microtentacles in detached HMLEs. Mechanistically, EMT downregulates the tubulin tyrosine ligase enzyme, resulting in an accumulation of detyrosinated α-tubulin (Glu-tubulin), and increases microtentacles that penetrate endothelial layers to facilitate tumor cell reattachment. Confocal microscopy shows that microtentacles are capable of penetrating the junctions between endothelial cells. Suppression of endogenous Twist in metastatic human breast tumor cells is capable of reducing both tubulin detyrosination and microtentacles. Clinical breast tumor samples display high concordance between Glu-tubulin and Twist expression levels, emphasizing the coupling between EMT and tubulin detyrosination in vivo. Coordinated elevation of Twist and Glu-tubulin at invasive tumor fronts, particularly within ductal carcinoma in situ samples, establishes that EMT-induced tubulin detyrosination occurs at the earliest stages of tumor invasion. These data support a novel model where the EMT that occurs during tumor invasion downregulates tubulin tyrosine ligase, increasing α-tubulin detyrosination and promoting microtentacles that could enhance the reattachment of circulating tumor cells to the vascular endothelium during metastasis.

Figure 1. EMT promotes increased McTNs and tubulin detyrosination in detached HMLEs

A: Phase contrast images of attached HMLE Twist and Snail cells compared to HMLE-GFP (top panel). Detached HMLE Twist and Snail display extensive membrane McTNs (lower panel, black arrows). B: Populations of live, detached HMLE Twist and Snail cells display significantly higher McTN frequencies than HMLE-GFP cells. Columns, mean for six experiments, in which at least 100 CellMask stained cells were counted; bars, SD. (P ≤ 0.005, t-test, black asterisks). C: Comparative expression profile of HMLE (GFP Vector Ctl, Twist, Snail) including E-cadherin, N-cadherin, vimentin, Twist, Snail, and β-actin as a loading control showing mesenchymal hallmarks in Twist and Snail HMLEs. D: HMLE Twist and Snail have increased Glu-tubulin levels and decreased levels of tubulin tyrosine ligase (TTL) compared to the HMLE-GFP while total α-tubulin and actin are comparable.

Figure 2. Glu-tubulin accumulation and reorganization during EMT

A: Attached HMLE-GFP ctl shows weak Glu staining while the HMLE Twist and Snail display filamentous accumulation and bundling of Glu-tubulin (arrows). Hoescht (d–f) was used to visualize nuclei. B: Detached cells were fixed and spun onto glass coverslips. Immunostaining indicates Glu-tubulin localization in HMLE Twist and Snail McTNs (white arrows).

Figure 3. Endogenous Twist suppression in MDA-MB-157s decreases Glu expression and organization

A: Transfection of a non-silencing control (NS ctl) or Twist siRNA (siTwist) in MDA-MB-157s shows suppression of Twist resulted in decreased Glu-tubulin levels. Vimentin and N-cadherin remained unchanged and E-cadherin repression persisted. HMLE-GFP was used as a positive control for E-cadherin expression and β-actin was used as a loading control. B: Following Twist suppression in MDA-MB-157s, nuclear staining of Twist (a,d) is decreased and delocalized compared to the NS Ctl. Twist suppression shows decreased and disrupted Glu (b,e) organization while vimentin (c,f) remained relatively unaffected. C: Live, detached MDA-MB-157s scored blindly following siTwist transfection displayed significantly lower McTN frequencies than NS ctl cells. Columns, mean for six experiments, in which at least 100 CellMask stained cells were counted; bars, SD. (P ≤ 0.05, T-test, black asterisks).

Figure 4. Immunohistochemical staining shows high concordance between Twist and Glu expression in patient tumor samples (33 patients; 66 samples)

A: Duplicate tissue microarrays stained for Glu and Twist were scored by intensity (negative, 0; weak, 1; moderate, 2; and strong, 3). Normal breast epithelial ducts (N) stain weakly for both Twist and Glu while the cancerous region (C) stains strongly (a,d). Patients with invasive ductal carcinoma exhibited high concordance (95%) between staining intensity and localization of Twist and Glu expression (b,e; white arrows). Discordance was observed in only 5% of patient samples (c,f; black arrows). Representative immunohistochemical scores are displayed in upper right corner. B: Low magnification shows an increase in staining intensity of Twist and Glu at the invasive front (IF) compared to the tumor center (TC) (a,b). Higher magnification shows stronger staining of Twist and Glu at the invasive front (b,e) while the tumor center stains weaker (c,f). C: Ductal carcinoma in situ (DCIS) samples (a,b) show weak expression of Twist and Glu in the tumor center enclosed by basement membrane (white arrows), but that both Twist and Glu are coordinately upregulated at sites of tumor invasion where the basement membrane is compromised (black arrows). Higher magnification image pairs (b,e and c,f) show Twist and Glu are upregulated in matching cells (black arrows).

Figure 5. HMLE Twist and Snail have increased reattachment rates

A: HMLE Twist () and Snail () lines attached at significantly faster rates then the HMLE-GFP (), as gauged by electrical impedence expressed as increasing cell index (CI). Lines, mean for 3 triplicate wells; bars, SD; representative graph shown. Three independent experiments were performed (Figure S4). B: CalceinAM-labeled HMLE Twist and Snail cells attach significantly more to confluent human bone marrow endothelial layers (HBME) at 1h than the HMLE-GFP cells (P≤0.005; T-test; black asterisks). Columns, mean for eight experiments; bars, SD. C) Representative images of calcein-AM labeled HMLEs over a confluent HBME layers.

Figure 6. Microtentacles facilitate HMLE-endothelial attachment

A: Confocal imaging of GFP-Membrane transfected HMLE cells suspended for 20min over a confluent layer of mCherry-labeled HBME. Top, angle, and side views of HMLE cells at the early stages of attachment show HMLE-GFP vector control cells rounded without observable McTNs. HMLE-Twist displays a McTN anchoring to the top of the HBME (white arrow). HMLE-Snail exhibits McTNs extending under (white arrow) or bending towards (white arrowheads) the HBME layer.