α-Tubulin acetylation elevated in metastatic and basal-like breast cancer cells promotes microtentacle formation, adhesion, and invasive migration

Abstract

Metastatic cases of breast cancer pose the primary challenge in clinical management of this disease, demanding the identification of effective therapeutic strategies that remain wanting. In this study, we report that elevated levels of α-tubulin acetylation are a sufficient cause of metastatic potential in breast cancer. In suspended cell culture conditions, metastatic breast cancer cells exhibited high α-tubulin acetylation levels that extended along microtentacle (McTN) protrusions. Mutation of the acetylation site on α-tubulin and enzymatic modulation of this posttranslational modification exerted a significant impact on McTN frequency and the reattachment of suspended tumor cells. Reducing α-tubulin acetylation significantly inhibited migration but did not affect proliferation. In an analysis of more than 140 matched primary and metastatic tumors from patients, we found that acetylation was maintained and in many cases increased in lymph node metastases compared with primary tumors. Proteomic analysis of an independent cohort of more than 390 patient specimens further documented the relationship between increased α-tubulin acetylation and the aggressive behaviors of basal-like breast cancers, with a trend toward increased risk of disease progression and death in patients with high-intensity α-tubulin acetylation in primary tumors. Taken together, our results identify a tight correlation between acetylated α-tubulin levels and aggressive metastatic behavior in breast cancer, with potential implications for the definition of a simple prognostic biomarker in patients with breast cancer.

Figure 1. Metastatic breast tumor cell lines have high acetylation of α-tubulin that is enriched in more numerous McTNs

(A) Lysates from non-metastatic (Non-Met.) and metastatic (Met.) breast cancer cell lines were subjected to immunoblot analysis for α-tubulin post-translational modifications. (B) Densitometric analysis of acetylated α-tubulin, as compared to total α-tubulin. Error bars indicate +/− standard deviation, n=3. Non-Met. vs. Met. breast tumor cells: **p<0.01 (C) Immunofluorescence for DNA (blue), α-tubulin (green), and acetylated α-tubulin (red). Scale bar=20 μm. (D) McTN counts, with data represented as the mean +/− standard deviation of n=3 experiments with at least 100 cells scored blindly in each independent experiment. **p<0.01 between Non-Met. and Met. McTN counts. (E) Representative suspended-cell images highlighting McTN protrusions (arrows). (F) Suspended cell immunofluorescence indicates acetylated α-tubulin (red) localizes along α-tubulin (green)-based protrusions in metastatic cells. DNA is stained in blue. Arrows indicate McTNs. Scale bar=10 μm.

Figure 2. K40R α-tubulin mutant decreases acetylation and McTN frequency

(A) MDA-MB-231 and BT-549 cells were transiently transfected with GFP-labeled K40R α-tubulin (arrows, top row) or GFP α-tubulin control (arrows, bottom row) and subjected to immunofluorescence for acetylated α-tubulin (red). DNA is stained in blue. Arrowheads indicate non-transfected cells. Scale bar=20 μm. (B) Stable cell lysates were taken of both attached (Att.) and suspended (Susp.) cells expressing the K40R α-tubulin mutant or wild-type (WT) α-tubulin control and subjected to immunoblot. (C) Representative McTN images of each stable cell line suspended for 30min under low-attach conditions. Arrows indicate McTNs. Scale bar=10 μm. (D) McTN counts were carried out on suspended stable cell lines. Error bars indicate +/− standard deviation of n=3 in triplicate. **p<0.01.

Figure 3. αTAT1 significantly increases α-tubulin acetylation and McTNs

(A) MCF-7 cells were transiently transfected with αTAT1-GFP or GFP control and subjected to immunofluorescence for acetylated α-tubulin (red). Transfected cells are indicated by arrows; non-transfected cells are indicated by arrowheads. DNA is stained in blue. Scale bar=20 μm. (B) Immunoblot of cells lysed after 24h transfection under attached or 30min suspended conditions. (C) McTN counts of MCF-7 cells transiently transfected with αTAT1-GFP or GFP control. Data represents n=3 in triplicate +/− standard deviation. **p<0.01. (D) Cells were subjected to suspended cell immunofluorescence for acetylated α-tubulin (red). Arrows indicate McTNs. Scale bar=10 μm.

Figure 4. α-tubulin acetylation significantly affects reattachment rates of suspended breast tumor cells

(A–C) Real-time cell reattachment was analyzed using the xCELLigence system. Each cell line was plated in triplicate and error bars indicate +/− standard deviation of those 3 wells. The graphs shown are representative of n=3 independent runs/cell line. Cell Index represents the change in electrical impedance over time. (A and B) Representative graphs of suspended cell reattachment for stable pooled clones expressing the mutant K40R α-tubulin or the α-tubulin wild-type control. (C) Representative graph of MCF-7 cells transiently transfected with αTAT1-GFP or GFP control 24h prior to analysis.

Figure 5. Chemotactic movement of breast tumor cells is affected by reducing acetylated α-tubulin

(A–C) Real-time migration was analyzed with the xCELLigence system. Modified electronic chamber plates (CIM-plates) were utilized. Each cell line was plated in triplicate into serum-free media (upper chamber) and allowed to migrate towards the lower chamber (containing 5% FBS) for 24h. The serum-free control represents control cells plated in duplicate into serum-free media (upper chamber) where the lower chamber also contained serum-free media. Error bars indicate +/− standard deviation of the 3 experimental or 2 control wells/run. Graphs shown are representative of n=3 independent runs/cell line. Cell Index represents the change in electrical impedance representing movement from the upper to lower chamber. (A and B) Representative chemotaxis graphs of MDA-MB-231 or BT-549 stable pooled clones expressing the K40R α-tubulin mutant or the α-tubulin control and (C) MCF-7 cells transiently transfected with αTAT1-GFP or GFP control. The underside of the upper chamber was stained at 24h and representative images are shown to the right of each graph.

Figure 6. Acetylated α-tubulin is detected in patient primary and matched metastatic tumors

Representative images of matched patient primary invasive ductal carcinomas and lymph node metastases stained for acetylated α-tubulin. Horizontal pairings represent the primary tumor and the matched lymphatic metastasis from the same patient. 8x magnification image contains the tumor score (scale bar=200 μm) and is compared to 20x of the same sample (scale bar=50 μm).

Figure 7. High acetylation of α-tubulin in patient primary tumors is linked to the basal-like subtype and an increased risk of disease progression and death

(A) Percentage of patients in each subtype with low or high acetylated α-tubulin intensity in the patient primary tumor. Acetylation was considered “Low” if it was below and “High” if it was above the median acetylation of n=392 patients. Basal (n=83), HER2 (n=48), luminal A (LumA) (n=168), and luminal B (LumB) (n=93). (B) Raw intensity values for acetylated α-tubulin in all patient primary tumors are compared by subtype. ***p<0.001. (C) Kaplan-Meier progression-free survival (PFS) and (D) overall survival (OS) for n=123 non-HER2 patients in the high and low acetylation categories. *p<0.05, HR= hazard ratio.