The combinatorial activation of the PI3K and Ras/MAPK pathways is sufficient for aggressive tumor formation, while individual pathway activation supports cell persistence.

A high proportion of human tumors maintain activation of both the PI3K and Ras/MAPK pathways. In basal-like breast cancer (BBC), PTEN expression is decreased/lost in over 50% of cases, leading to aberrant activation of the PI3K pathway. Additionally, BBC cell lines and tumor models have been shown to exhibit an oncogenic Ras-like gene transcriptional signature, indicating activation of the Ras/MAPK pathway. To directly test how the PI3K and Ras/MAPK pathways contribute to tumorigenesis, we deleted PTEN and activated KRas within non-tumorigenic MCF-10A breast cells. Neither individual mutation was sufficient to promote tumorigenesis, but the combination promoted robust tumor growth in mice. However, in vivo bioluminescence reveals that each mutation has the ability to promote a persistent phenotype. Inherent in the concept of tumor cell dormancy, a stage in which residual disease is present but remains asymptomatic, viable cells with each individual mutation can persist in vivo during a period of latency. The persistent cells were excised from the mice and showed increased levels of the cell cycle arrest proteins p21 and p27 compared to the aggressively growing PTEN-/-KRAS(G12V) cells. Additionally, when these persistent cells were placed into growth-promoting conditions, they were able to re-enter the cell cycle and proliferate. These results highlight the potential for either PTEN loss or KRAS activation to promote cell survival in vivo, and the unique ability of the combined mutations to yield rapid tumor growth. This could have important implications in determining recurrence risk and disease progression in tumor subtypes where these mutations are common.

ROCK inhibition promotes microtentacles that enhance reattachment of breast cancer cells.

The presence of circulating tumor cells (CTCs) in blood predicts poor patient outcome and CTC frequency is correlated with higher risk of metastasis. Recently discovered, novel microtubule-based structures, microtentacles, can enhance reattachment of CTCs to the vasculature. Microtentacles are highly dynamic membrane protrusions formed in detached cells and occur when physical forces generated by the outwardly expanding microtubules overcome the contractile force of the actin cortex. Rho-associated kinase (ROCK) is a major regulator of actomyosin contractility and Rho/ROCK over-activation is implicated in tumor metastasis. ROCK inhibitors are gaining popularity as potential cancer therapeutics based on their success in reducing adherent tumor cell migration and invasion. However, the effect of ROCK inhibition on detached cells in circulation is largely unknown. In this study, we use breast tumor cells in suspension to mimic detached CTCs and show that destabilizing the actin cortex through ROCK inhibition in suspended cells promotes the formation of microtentacles and enhances reattachment of cells from suspension. Conversely, increasing actomyosin contraction by Rho over-activation reduces microtentacle frequency and reattachment. Although ROCK inhibitors may be effective in reducing adherent tumor cell behavior, our results indicate that they could inadvertently increase metastatic potential of non-adherent CTCs by increasing their reattachment efficacy.

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

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.

Curcumin targets breast cancer stem-like cells with microtentacles that persist in mammospheres and promote reattachment.

Cancer stem-like cells (CSC) and circulating tumor cells (CTC) have related properties associated with distant metastasis, but the mechanisms through which CSCs promote metastasis are unclear. In this study, we report that breast cancer cell lines with more stem-like properties display higher levels of microtentacles (McTN), a type of tubulin-based protrusion of the plasma cell membrane that forms on detached or suspended cells and aid in cell reattachment. We hypothesized that CSCs with large numbers of McTNs would more efficiently attach to distant tissues, promoting metastatic efficiency. The naturally occurring stem-like subpopulation of the human mammary epithelial (HMLE) cell line presents increased McTNs compared with its isogenic non-stem-like subpopulation. This increase was supported by elevated α-tubulin detyrosination and vimentin protein levels and organization. Increased McTNs in stem-like HMLEs promoted a faster initial reattachment of suspended cells that was inhibited by the tubulin-directed drug, colchicine, confirming a functional role for McTNs in stem cell reattachment. Moreover, live-cell confocal microscopy showed that McTNs persist in breast stem cell mammospheres as flexible, motile protrusions on the surface of the mammosphere. Although exposed to the environment, they also function as extensions between adjacent cells along cell-cell junctions. We found that treatment with the breast CSC-targeting compound curcumin rapidly extinguished McTN in breast CSC, preventing reattachment from suspension. Together, our results support a model in which breast CSCs with cytoskeletal alterations that promote McTNs can mediate attachment and metastasis but might be targeted by curcumin as an antimetastatic strategy.

Parthenolide and costunolide reduce microtentacles and tumor cell attachment by selectively targeting detyrosinated tubulin independent from NF-κB inhibition.

INTRODUCTION: Detyrosinated tubulin, a post-translational modification of α-tubulin and a hallmark of stable microtubules, has gained recent attention given its association with tumor progression, invasiveness, and chemoresistance. We also recently reported that epithelial-to-mesenchymal transition (EMT) promotes tubulin detyrosination through tubulin tyrosine ligase (TTL) suppression. Furthermore, detyrosinated tubulin-enriched membrane protrusions, termed microtentacles (McTN), facilitate tumor cell reattachment to endothelial layers. Given the induction of EMT associated with inflammation and cancer progression, we tested anti-inflammatory nuclear factor-kappaB (NF-κB) inhibitors on a panel of human breast carcinoma cells to examine their effects on detyrosinated tubulin to identify more specific tubulin-directed anti-cancer treatments. METHODS: Using metastatic human breast carcinoma cells MDA-MB-157, MDA-MB-436, and Bt-549, we measured the impact of NF-κB inhibitors parthenolide, costunolide, and resveratrol on detyrosinated tubulin using protein expression analysis and immunofluorescence. A luciferase reporter assay and a viability screen were performed to determine if the effects were associated with their NF-κB inhibitory properties or were a result of apoptosis. Real-time monitoring of cell-substratum attachment was measured utilizing electrical impedance across microelectronic sensor arrays. We compared the selectivity of the NF-κB inhibitors to specifically target detyrosinated tubulin with traditional tubulin-targeted therapeutics, paclitaxel and colchicine, throughout the study. RESULTS: Sesquiterpene lactones, parthenolide and costunolide, selectively decrease detyrosinated tubulin independent of their inhibition of NF-κB. Live-cell scoring of suspended cells treated with parthenolide and costunolide show reduction in the frequency of microtentacles and inhibition of reattachment. Structural analysis shows that parthenolide and costunolide can decrease detyrosinated microtubules without significantly disrupting the overall microtubule network or cell viability. Paclitaxel and colchicine display indiscriminate disruption of the microtubule network. CONCLUSIONS: Our data demonstrate that selective targeting of detyrosinated tubulin with parthenolide and costunolide can reduce McTN frequency and inhibit tumor cell reattachment. These actions are independent of their effects on NF-κB inhibition presenting a novel anti-cancer property and therapeutic opportunity to selectively target a stable subset of microtubules in circulating tumor cells to reduce metastatic potential with less toxicity in breast cancer patients.