Intracellular reduction in ATP levels contributes to CYT997‐induced suppression of metastasis of head and neck squamous carcinoma

Abstract The incidence rate of head and neck squamous cell carcinoma (HNSCC) has steadily increased over the past decade. However, treatment options for metastatic HNSCC are often limited and the 5‐year survival rate has remained static. Therefore, the development and assessment of more efficient but less toxic therapeutic strategies is an unmet need for treatment of more extensive HNSCC. Here, we report that CYT997, a novel microtubule‐disrupting agent, exerts strong activity in inhibiting HNSCC cell invasion and metastasis. The loss of invasion capacity by CYT997 was accompanied by an associated increase in cell adhesion and the reversal of epithelial‐mesenchymal transition (EMT). Increased expression of E‐cadherin protein and decreased expression of Vimentin protein became evident in HNSCC cells following CYT997 exposure, which were consistently observed in HNSCC xenografts from the mice receiving CYT997. Moreover, the capacity of invasive HNSCC cells to form pulmonary metastases was significantly blocked with CYT997 treatment, indicating that the diminishment of EMT traits contributes to CYT997‐suppressed metastasis. Intriguingly, CYT997 impaired intracellular ATP levels in HNSCC cells, at least in part, through its inhibitory effect on the mitochondrial protein IF1. The addition of ATP attenuated CYT997‐induced suppression of cell invasion, coupled with down‐regulation of E‐Cadherin and up‐regulation of Vimentin. These findings support a critical role of ATP levels in cell invasion and metastasis under the influence of CYT997. Collectively, our data unveil the mechanism involved in mediating CYT997 action, and provide preclinical rationale for possible clinical application of CYT997 as a novel therapeutic strategy against aggressive HNSCC.

strategies against HNSCC metastasis. Generally, patients with early stage HNSCC, particularly those limited to the site of origin, are treated with either surgery or radiation therapy. If patients have more extensive cancers, they often receive chemotherapy. However, treatment options for recurrent and metastatic HNSCC are often limited, with palliative treatment generally offered in these situations. Therefore, the development and assessment of a new chemotherapy, which holds great potential for higher efficacy and lesser side effects, are imperative for the management of HNSCC, especially for metastatic tumours.
Microtubules are filamentous intracellular structures that are responsible for the maintenance of cell shape and the facilitation of cell motility. 3 Targeting microtubules to disrupt their normal function within the cancer cell has proven to be one of the best classes of cancer chemotherapeutic drugs available in clinics to date. 4 The microtubule-targeting agents (MTAs), including Paclitaxel and Docetaxel, are very effective cancer drugs with therapeutic benefits in both hematopoietic and solid tumours. These drugs, as "stabilizers" or "destabilizers" of microtubules, potently suppress the dynamic stability of the microtubules to block mitotic progression and trigger apoptosis. 4,5 However, there is little evidence how MTAs affect cell motility, especially in the process of cancer cell invasion and metastasis.
CYT997, a new microtubule-disrupting agent inhibiting tubulin polymerization and disrupting cellular microtubules, has been used as an anticancer chemo drug for a wide range of cancer types. [6][7][8] The oral activity of CYT997 is more effective than most other MTAs.
Phase I clinical trials have assessed the anticancer efficacy and safety of CYT997 in patients with solid tumours, and Phase II clinical trials are performing studies for the treatment of selected tumors. 8,9 We have found that CYT997 can trigger oxidative stress-associated apoptosis and mTOR-dependent autophagy in HNSCC cells, and CYT997-induced autophagy appears to have a protective role against apoptosis by inhibiting the induction of excessively high reactive oxygen species (ROS). 10 A superior inhibitory effect of CYT997 on prostate tumour growth and metastasis was demonstrated in our previous work, suggesting that CYT997 may also regulate the potential of invasion and metastasis of HNSCC cells.
Here, we study the anticancer activity of CYT997 in cell invasion and metastasis using cultured HNSCC cell lines and in vivo animal models. We show that CYT997 suppresses invasion and metastasis of HNSCC cells by increasing cell adhesion and reversing epithelialmesenchymal transition (EMT), which largely depends on the reduction in intracellular levels of ATP production. By testing the effects of the addition of ATP on CYT997-induced suppression of invasion, we provide evidence that intracellular reduction in ATP levels has an important role in limiting HNSCC cell invasion/metastasis under the influence of CYT997.

| Cell lines and culture conditions
Invasive HN6 and HN12 cells derived from HNSCCs have been previously described. 11  2.4 | Three-dimensional (3D) tumour spheroid invasion assays 3D invasion assays were performed as we previously described. 16 Briefly, 2 × 10 4 cells were incubated overnight to form 3D spheroid in hanging droplet in a well of an inverted round bottom 96-well plate, followed by adding150 μl mixture of Matrigel: DMEM without serum at the ratio 1:1. Then 150 μl complete culture medium containing double doses of CYT997 were added. After 4 days, invaded cells from spheroids were imaged under a microscope.

| Mitochondrial staining
MitoTracker™ Red CMXRos obtained from Invitrogen (Carlsbad, CA) was used to label mitochondria within live cells utilizing the mitochondrial membrane potential according to manufacturer's instruction. The intensity of MitoTracker was measured by fluorescence microscope and flow cytometer. All flow cytometer data were analysed using FlowJo software (Tree Star, Ashland, OR).

| Adhesion assays
The binding ability of cells to components of the extracellular matrix was determined by CytoSelect™ 48-Well Cell Adhesion Assay (Cell ZHAO ET AL.

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Biolabs, San Diego, CA). Briefly, 1 × 10 5 cells with or without CYT997 treatment were plated onto extracellular matrix-coated wells and incubated for 90 minutes at 37°C under 5% CO 2 . Medium was removed, and cells were washed with PBS before the addition of Cell Stain Solution. The cells were then washed and dried, followed by the incubation of Extraction Solution for 10 minutes. The extracted samples were transferred to a 96-well plate and the optical density measured at 560 nm.

| Animal studies
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Augusta University. Sixweek-old NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). To generate a xenotransplantation model, exponentially growing 1 × 10 6 HN12 cells were suspended in 100 μl PBS and intravenously injected into tail vein of NSG mice. The tumour-bearing mice were randomized (n = 5/group) for different treatment with vehicle and 20 mg/kg CYT997 daily by oral gavage respectively. The mice were sacrificed on treatment day 42, and the xenografts and lungs were removed and processed for Western blot and histopathological analysis.

| Statistical analysis
Statistical analyses were performed by unpaired Student's t test for two group comparisons and ANOVA for multigroup comparisons at a significance level of P < 0.05. Where indicated, the results were representative of at least three independent experiments performed in triplicate and were expressed as the mean ± SD.

| CYT997 suppresses migration and invasion in HNSCC cells
Our previous study has shown that CYT997 can inhibit cell viability and induce oxidative stress-associated apoptosis in HNSCC cells. 10 These data prompted us to investigate whether CYT997 has other anticancer potential in HNSCC cells. To mitigate the inhibitory effect on cell viability, we used 20 nmol/L CYT997 in the following in vitro studies, which was much lower than IC50 of 100 nmol/L in HNSCC cells. HN6 and HN12 are two highly invasive HNSCC cell lines, and no significant growth rate and cell death was noted in these cells, in the presence or absence of 20 nmol/L CYT997 (data not shown). However, CYT997 treatment led to a clear reduction in wound-healing capability ( Figure 1A).
Matrigel invasion assays further showed reduced invasion potential in HN6 and HN12 cells following exposure of CYT997 ( Figure 1B).
To determine the contribution of EMT in CYT997-induced suppression of cell invasion, we assessed the morphological changes of cells with or without CYT997 treatment. Long-term treatment (3 days) of HN6 and HN12 cells with CYT997 reversed EMT as evidenced by converting from an elongated mesenchymal shape into a "cuboidal" epithelial structure ( Figure 1C). Moreover, 3D tumour spheroid invasion followed over a period of 4 days as shown in Figure 1D revealed that invasion from HN6-and HN12-derived spheroids was much less pronounced in CYT997 treatment compared with the control groups treated with DMSO.

| CYT997 inhibits HNSCC cell EMT
To understand the mechanism underpinning CYT997-induced suppression of invasion, we sought to determine the molecules mostly   Figure 3D, E). These data indicate that CYT997 suppresses metastasis, at least in part, through inhibiting EMT of HNSCC cells.

| CYT997 induces mitochondrial dysfunction and impairs intracellular ATP levels in HNSCC cells
Our work has demonstrated that CYT997 can induce oxidative stress in HNSCC cells as evidenced by elevated ROS levels and enhanced superoxide release, 10 Figure 4E). IF2 was only increased in HN12 cells following drug exposure, suggesting CYT997-induced alterations in this protein is cell-content dependent ( Figure 4E). Other proteins examined in this study, including Catalase, HSP60, DRP1, PTPMT1, Cytochrome C, and ATPAF1, have not shown notable changes in their levels with or without CYT997 treatment ( Figure 4E).

Most of the ATP in cells is produced by ATP synthase in mito-
chondria, and IF1 is a natural inhibitor protein of mitochondrial ATP synthase. 18 Given the fact that CTY997 induced repression

| Restoration of ATP attenuates CYT997induced suppression of invasion in HNSCC cells
The maintenance of an adequate ATP supply is of crucial importance for the mechanisms of structural remodelling in cells with high shape plasticity. 19 Therefore, we determined whether the loss of ATP played an essential role in CYT997-induced suppression of cells invasion. CYT997 treatment alone appeared no effect on viability in both HN6 and HN12 cells, and addition of ATP to CYT997 did not change this phenotype ( Figure 5A). MitoTracker staining showed the control cells displayed mitochondria that stained brightly and were a mixed reticulum with tubular and round form, whereas in the presence of CYT997, cells exhibited more round or fragmented mitochondria ( Figure 5B). Addition of ATP did not rescue the changes in mitochondrial morphology induced by CYT997 ( Figure 5B). However, increased invading cells were observed in both CYT997-treated cells upon ATP administration ( Figure 5C Oral drug administration is the most convenient method of medication and now more than 60% of marketed drugs are used as oral products. CYT997 can be well absorbed after oral administration, with mean Tmax and T1/2 of 1.1 and 3.2 hours, respectively. 9 In our previous animal study, tumour-bearing mice treated with CYT997 by oral gavage showed significantly smaller xenografts compared with control mice. 10 We have not successfully identified metastatic HNSCC cells in the lungs of mice in the previous study, which may be due to the termination of the animal experiment too early. In this study, we inoculated a low number of HN12 cells (5-fold cells less than the last experiment) into the flank of NSG mice and extended the experimental window for three more weeks, allowing tumour cells to develop metastatic tumours before the tumour burden became excessive. It is clear that, in the absence of CYT997, highly invasive HN12 cells can metastasize to the lung from a primary site in NSG mice. In contrast, much fewer cancer cells can be observed in the lungs of the mice following CYT997 treatment, indicating that CYT997 has a superior anticancer activity by inhibiting tumour growth and metastasis, coincidently. Recently, an orthotopic mouse model of HNSCC has been established by the implantation of cancer cells into the floor of a mouse's mouth, allowing us to better study the anticancer effects of CYT997. Pharmacokinetics and pharmacodynamics of this drug will be also beneficial from using an orthotopic mouse model, which paves the way to the successful, practical use of CYT997 in cancer treatment.
CYT997 is reported as an available antiangiogenic agent to suppress the local invasion and distant metastasis of malignant tumours, but whether CYT997 plays an importance in inhibition of EMT in cancer cells remains unclear. Here we reveal that CYT997 can induce significant repression of invasion and EMT phenotypes, which is abrogated by ATP treatment. It appears that CYT997 decreases ATP production and, in turn, up-regulates E-Cadherin and down-regulates Vimentin to facilitate the reversal of EMT, resulting in the decreased invasion and metastasis of HNSCC cells. Elevated ROS generation is engaged in CYT997-induced inhibition of cell viability. 10 We treated HN6 and HN12 cells with NAC, an antioxidant with the ability to minimize oxidative stress, in the presence or absence of CYT997. No significant changes in cell invasion and EMT were seen with or without NAC treatment (data not shown), excluding the role of ROS in cell movement-related phenotype in CYT997 treatment. It looks that, on one hand, CYT997-induced ROS is the key player involved in the enhancement of apoptosis and autophagy ( Figure 6), and increased autophagy by CYT997 plays a prosurvival role through inhibiting ROS-dependent apoptosis in HNSCC cells. 10 On the other hand, CYT997-induced blockade of ATP release suppresses HNSCC cell invasion and metastasis ( Figure 6). These As the "principal energy currency," ATP is involved in metabolism and most of the other cellular activities. 19 However, the role of intracellular ATP in sustaining cell motility is very obscure, which may depend on its levels and distribution pattern. Mitochondria is the site of ATP generation, and the dysfunction of mitochondria in HNSCC cells has been found in the presence of CYT997, which may explain how the cellular levels of ATP production were impaired.
Tumour microenvironment is ATP rich, suggesting a role for puriner- How ZO-3 is up-regulated by CYT997 is one of our follow-up investigations. In summary, we demonstrate that CYT997 strongly suppresses invasion and metastasis of HNSCC cells, which is accompanied by reduction in intracellular ATP production. The addition of ATP levels hampers the efficacy of CYT997 in inhibition of cancer invasion and metastasis, which suggests that blockage of ATP by CYT997 is one of the main mechanisms in understanding druginduced changes in cancer cell motility. Our findings provide a rationale to use CYT997 as an effective therapeutic strategy for either primary or metastatic HNSCC. F I G U R E 6 Schematic representation of drug mechanisms of CYT997 in HNSCC cells. In HNSCC cells, induction of ROS by CYT997 triggers autophagy to attenuate its mediated apoptosis, and blockade of ATP release by CYT997 suppresses cell invasion and metastasis