Metastatic breast tumors express increased tau, which promotes microtentacle formation and the reattachment of detached breast tumor cells

Abstract

The cytoskeletal organization of detached and circulating tumor cells (CTCs) is currently not well defined and may provide potential targets for new therapies to limit metastatic tumor spread. In vivo, CTCs reattach in distant tissues by a mechanism that is tubulin-dependent and suppressed by polymerized actin. The cytoskeletal mechanisms that promote reattachment of CTCs match exactly with the mechanisms supporting tubulin microtentacles (McTN), which we have recently identified in detached breast tumor cells. In this study, we aimed to investigate how McTN formation is affected by the microtubule-associated protein, tau, which is expressed in a subset of chemotherapy-resistant breast cancers. We demonstrate that endogenous tau protein localizes to McTNs and is both necessary and sufficient to promote McTN extension in detached breast tumor cells. Tau-induced McTNs increase reattachment of suspended cells and retention of CTCs in lung capillaries. Analysis of patient-matched primary and metastatic tumors reveals that 52% possess tau expression in metastases and 26% display significantly increased tau expression over disease progression. Tau enrichment in metastatic tumors and the ability of tau to promote tumor cell reattachment through McTN formation support a model in which tau-induced microtubule stabilization provides a selective advantage during tumor metastasis.

Figure 1. McTN formation increases with tau expression

(A) Western blots for tau (45–75kDa) revealed an increasing expression and molecular weight range as tumorigenicity increases from the HMEC cells to HCC1428. β-actin served as loading control. (B) McTN frequency increases concomitantly with tau expression. HCC1428 forms significantly more McTNs than lower tau expressing cells lines. ZR-75-1 forms significantly more McTNs than, HMEC, 184A1, 184B5, MCF-10A and Sk-Br-3. Data (n=3) are represented as the mean ± s.d. *P<0.05. (C) ZR-75-1 and HCC1428 were subjected to indirect IF for tau followed by direct IF for α-tubulin. As tau expression increases, localization of tau within McTNs increases (white arrows). Cells and McTNs displayed are representative of those under conditions of actin depolymerization.

Figure 2. Exogenous tau overexpression stimulates McTN formation

McTNs were evaluated blindly in live MCF-10A, Sk-Br-3, MCF-7, and BT-20 cells exogenously overexpressing pcDNA (left micrograph panels, white bars) or tau (right micrograph panels, black bars) in VC, LA, and Taxol+LA. Tau overexpression caused distinct morphological alterations in length, thickness and ridigity of McTNs formed compared to pcDNA. Tau-induced McTNs became longer and thicker in VC conditions compared to pcDNA. Treatment of tau-transfected cells with LA increased the length, thickness, and rigidity of McTNs comparable to empty vector cells treated with Taxol+LA. Frequency of McTN formation (lower graphs) revealed that tau significantly increased McTN formation. Data (n=3) are represented as the mean ± s.d. *P<0.05, scale bars = 10µm.

Figure 3. shRNA-mediated knockdown of tau in ZR-75-1 cells significantly reduces McTN formation

(A) siTau1 and siTau2 express less tau than the NSC parent line on Western blots. β-actin served as a loading control. (B) Under conditions of actin depolymerization with LA (black bars), siTau1 and siTau2 possess significantly less McTNs compared to NSC. When left untreated in VC (white bars), siTau2 form significantly less McTNs than NSC. Data (n=3) represents the mean ± s.d. *P<0.05; **P<0.001

Figure 4. Initial cell attachment is dependent on McTN formation as affected by tau

(A) Over 1h, MCF7 cells stably overexpressing GFP-tau (MCF-7tau1 and MCF-7tau2) attach more efficiently (200% and 180%, respectively) compared to MCF-7 controls. Data (n=4) represents the mean percentage of attachment ± s.d. *P<0.01. (B) Over 1h, siTau1 and siTau2 attach less efficiently (67% and 46%, respectively) compared to ZR-75-1 controls. Data (n=3) is represented as the mean percentage of attachment ± s.d. *P<0.01. (C) Glowscale of luciferase-expressing cells injected into the tail vein of nude mice demonstrate equal initial signal retention in regions of interest (ROI) emcompassing the lungs. Over a period of 8d post-injection, MCF-7tau1-luc cells are more retained than control MCF-7GFP-luc cells. (D). On average over all injections, 61.8% and 44.4% of MCF-7tau1-luc cells (n=6) were retained in lung capillaries 1d and 8d post-injection. In comparison, 15.9% and 12.9% of control MCF-7GFP-luc cells (n=5) were retained at the same time points. This difference was significantly different for both 1d (*P<0.02) and 8d (**P<0.05) post-injection by a non-parametric t-test.

Figure 5. Tau is expressed in primary breast tumors and matched lymph node metastases

Primary tumors and patient matched lymph node metastases displayed variable levels of tau expression. 0 = No expression, 1+ = low expression, and 2+–3+ = high expression. A number of lymph node metastases displayed tau expression despite no expression in matched primary tumors, while other patients displayed stable or increased expression in lymph node deposits compared to primary tumor. Large panels, scale bar = 125µm; Top small panels, scale bar = 63µm; Bottom small panels, scale bar = 31µm. Tau expression increases significantly in metastatic tumors (Table 1, p=0.0169).

Figure 6. Model of tau-induced McTN formation

In detached CTCs expressing tau, tau binds to and stabilizes microtubules, inducing microtubule bundling [1] and/or increasing individual polymer strength [2]. These microtubules are more able to penetrate the actin cortex due to preexisting defects in actin bundling or tau-induced actin depolymerization via priming of Src phosphorylation (Sharma et al., 2007). Consequently, microtubules easily deform the plasma membrane into McTNs that increase the ability of the cell to reattach to an ECM and improve metastatic efficiency. In detached CTCs lacking tau, microtubules are more dynamic and weaker; therefore, they are only able to form short McTNs [3] on none at all [4] due to the constriction of the actin cortex, even in the presence of preexisting cortical weaknesses. The lack of tau expression results in decreased efficiency of attachment and metastasis.