The toxic effect of cytostatics on primary cilia frequency and multiciliation

Abstract The primary cilium is considered as a key component of morphological cellular stability. However, cancer cells are notorious for lacking primary cilia in most cases, depending upon the tumour type. Previous reports have shown the effect of starvation and cytostatics on ciliogenesis in normal and cancer cells although with limited success, especially when concerning the latter. In this study, we evaluated the presence and frequency of primary cilia in breast fibroblasts and in triple‐negative breast cancer cells after treatment with cytostatics finding that, in the case of breast fibroblasts, primary cilia were detected at their highest incidence 72 hours after treatment with 120 nM doxorubicin. Further, multiciliated cells were also detected after treatment with 80 nM doxorubicin. On the other hand, treatment with taxol increased the number of ciliated cells only at low concentrations (1.25 and 3.25 nM) and did not induce multiciliation. Interestingly, triple‐negative breast cancer cells did not present primary cilia after treatment with either doxorubicin or taxol. This is the first study reporting the presence of multiple primary cilia in breast fibroblasts induced by doxorubicin. However, the null effect of these cytostatics on primary cilia incidence in the evaluated triple negative breast carcinomas cell lines requires further research.


| INTRODUC TI ON
Primary cilium, an organelle found on nearly every cell in the human body, typically serves as a mechanical sensory tool; further, it is also involved in cell proliferation and embryonic development. This organelle is dynamically regulated during the cell cycle, appearing during the G0/G1 phases and resorbed prior to mitosis. 1 The exact solubilization moment of primary cilia is determined by cell type and the expression of genes affecting resorption, such as Aurora A, Plk1 and TcTex-1. 2,3 Usually, a cell has only one primary cilium which, as previously mentioned, is involved in morphogenesis, cell proliferation and differentiation signalling. 4,5 Should there be a multiplication of centrosomes, a higher number of primary cilia will appear on the surface of cells as well, often bearing the same length and construction design, and spawning from the same ciliary pocket; however, the presence of multiple cilia has been mostly recorded in solid tumours 6,7 after exposure to ionizing irradiation 8,9 or in ciliopathies. 10 Typically, primary cilia are always observed in myoepithelial cells and fibroblasts and with low incidence in luminal epithelium cells.
Regarding cancer cells, a study of 26 breast cancer biopsy samples revealed the presence of primary cilia only on exceptional cases, especially in epithelial cells. 11 Basal subtype B tumours, a classification of triple negative breast carcinomas (TNBCs), are characterized by the absence of oestrogen, progesterone and Her2/neu receptors, 12 as well as by the rare presence of primary cilia. Among breast cancer tumours, TNBC has an estimated incidence of 10%-20%, although from a histological point of view TNBCs are little differentiated and are often included in basal-like subgroups. From a clinical standpoint, these tumours are frequently resistant to treatment, have quick progression, low 5-year survival rate, increased local recurrence and are highly metastatic.
This kind of tumours can be observed at any age; however, they mostly occur accompanied by BRCA1 mutations in younger women (>40 years of age). 13 Chemotherapy is the treatment of choice for triple-negative breast cancer patients, of which Doxorubicin and Taxol are the standard chemotherapeutic agents used as anticancer therapy in combination with α-HER2/neu receptor targeted therapy.
Doxorubicin belongs to the anthracyclines group, whereas Taxol is considered as a taxane. The former is an effective intercalating cytotoxic agent used in the treatment of various tumour types and commonly used in combination with the latter in adjuvant and neoadjuvant therapeutic strategies for breast cancer patients. 14 Previous research on the effect of cytostatics on primary cilia has focused in the effects of Taxol over the elongation and shortening of primary cilia. In a study by Sharma et al., Taxol was shown to block the emergence of primary cilia in mammalian cell cultures. 15 However, low concentrations result in an increased quantity of free tubulin subunits in the cytosol, leading to enlarged primary cilia. 16,17 Ongoing research highlights two important questions in the cell biology of cancer and primary cilia: (a) the significance of having primary cilia in normal cells and (b) the loss of primary cilia in cancer cells and its relation to drug resistance. 18 Therefore, the increased primary cilia frequency induced by cytostatics could be used in other studies trying to assess the toxicity of these drugs.

| Cell culture
Unless otherwise stated, all standard chemicals and antibodies were purchased from Sigma-Aldrich, Czech Republic. In this study,

| Cytostatic drugs
Doxorubicin and Taxol were dissolved in 0.5% DMSO and kept in 1 mM stock solutions. Doxorubicin and Taxol were diluted in culture media before use at a ratio of 1:100 and 1:1000 respectively.

| Cell treatment and immunofluorescence
MDA-MB-231, BT-549 and fibroblasts cells were cultured in 6-well plates at a density of 3 × 10 5 cells per well and incubated at 37°C/5% CO 2 for 24 hours, each well contained a gelatine-coated coverslip. After this period, the cells were treated with Doxorubicin (10, 20, 40, 80, and 120 nM) or Taxol (1.25

| Transmission electron microscopy (TEM)
Fibroblasts were fixed in 3% glutaraldehyde (in 0.1 M cacodylate buffer, pH 7.2) for 5 minutes at 37°C and then for 3 hours at room tem-

| Statistical analysis
Graphs were made using the GraphPad Prism 6 biostatistics software (GraphPad Software, USA). The statistical analysis between the evaluated groups was performed with one-way ANOVA followed by a post-hoc Tukey test (P < 0.05).

| Primary cilia incidence and multiciliation induced by the cytostatics Doxorubicin and Taxol
Regarding Doxorubicin, the number of fibroblasts with primary cilia increased to ~70%, in comparison with control untreated cells, after 72 hours of treatment with various concentrations of this cytostatic (10, 20, 40, 80 and 120 nM), observing a higher incidence after treatment with 120 nM Doxorubicin. Overall, this effect could be observed evenly across the entire dose range used in this study (Figure 2A). After 72 hours of treatment, primary cilia were detected by immunostaining ( Figure 2B -control cells; Figure 2C -120 nM Doxorubicin) and electron microscopy ( Figure 2D  for 72 hours, an effect that was repeated in BT-549 cells ( Figure 5).

| D ISCUSS I ON
In this work, we sought to determine the effect that the cytostatics Doxorubicin and Taxol could have on primary cilia incidence in triple-negative breast cancer cells. We chose these cytostatics because they are commonly used in the treatment of breast cancer.
On one hand, Doxorubicin (DOX) acts by intercalating into the DNA strands inhibiting topoisomerase II activity and thus inducing strand breaks when DNA is being replicated; in addition, it also promotes the formation of reactive oxygen species (ROS), which are highly toxic. Taxol, on the other hand, inhibits microtubule depolymerisation, 19 resulting in shorter primary cilia and affecting their frequency in the exposed cells 15 ; therefore, it was included as a negative-control drug in our experiments.
Solid tumours possess a characteristic absence or low incidence of primary cilia which, when present, may also have a compromised structure and function. However, some tumour types that are dependent upon the Hedgehog (Hh) signalling pathway often have an increased frequency of primary cilia. 20,21 Further, several types of tumours have been associated with altered Hh, Wnt, NOTCH and Hippo signalling pathways, which are related to primary cilia; therefore, compromised signal transduction could also be caused by defects in the formation, structure or function of primary cilia. 22 Primary cilia normally occur in approximately 70% of fibroblasts and from 7% to 19% of epithelial cells from healthy breast tissue. 11 However, the study of 11 breast cancer cell lines revealed that primary cilia were only present in four of these and at the very low frequency of 0.3%-4%; curiously, these cell lines had the shared characteristic of being basal B subtypes, which are analogous to triple-negative breast cancer cells. 11 Regardless, primary cilia have been found in some cases of TNBCs, which hints at the existence of several TNBC subtypes in which the reason for this exclusive presence of primary cilia is yet unclear. A possible explanation could be that these tumour subtypes are dependent upon the Hh signalling pathway and hence upon primary cilia, as it has been observed in most basal cell carcinomas and medulloblastomas. 22 In this study, the chemosensitive triple-negative breast cancer cell lines BT-549 and MDA-MB-231 were used, in addition to normal skin fibroblasts as a comparative control, to test the effect that these cytostatics could have on primary cilia incidence. The cell lines were 20% to 40% of fibroblasts showed two or more cilia after treatment with doses of 20-120 nM, reaching the maximum value of 40% multiciliated cells after treatment with 80 nM. It must be mentioned that multiciliated cells were also in possession of multiple centrosomes, an effect that has also been observed in some tumour cells after exposure to cytostatics or ionizing radiation. 7,9 The treatment with Taxol also resulted in increased primary cilia frequency, reaching a value of 80% after 72 hours of treatment with 1.25 and 3.25 nM; however, the exposure to higher doses Based on previous results we were aware that primary cilia are absent or in very low percentage in the triple-negative breast cancer cell lines BT-549 and MDA-MB-231. 11,24,25 This absence of primary cilia has been associated with a loss of function mutation in the tumour suppressor gene p53. 26 However, the effect that Doxorubicin could have on primary cilia frequency in these cell lines has been heretofore unreported. Primary cilia play an important role in cell growth and tissue homeostasis and, in normal cells, the development of primary cilia is a dynamic process whose formation can occur in either G0/G1 or, more commonly, during the S/G2 phases; however, and almost without exception, the cilium is absorbed before entering mitosis and reappears once again in post-cytokinetic phases of the cell cycle.
This periodic cilium absorption is therefore related to the cell cycle and affects cell sensitivity to external signals associated with cilia receptors. 32 TNBC cells, however, are notorious for their lack of primary cilia, although they can sometimes occur with extremely low frequency. 33 Regardless, this low-primary cilium frequency cannot be increased under serum starvation conditions in TNBC cells, which has been a proven method in healthy cells. 11 Our study reports the null effect of Doxorubicin or Taxol on the incidence of primary cilia in triple-negative breast cancer cell lines where. Further, we also report the presence of multiple primary cilia in breast fibroblasts induced by Doxorubicin which, to the best of our knowledge, is now reported for the first time. The null effect of these cytostatics on primary cilia incidence in the evaluated TNBC cell lines, as opposed to their effect on healthy cells, hints at some larger mechanism at play that could be involved with the inherent characteristics of malignant cells; however, these considerations must be addressed further and more in depth before an accurate conclusion can be reached. for his kind assistance in English language revision.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.