2022 Volume 45 Issue 11 Pages 1627-1635
Eribulin, an inhibitor of microtubule dynamics, is used for treating breast cancers and sarcomas. The microtubule-destabilizing protein stathmin may modulate the antiproliferative activity of eribulin on breast cancer cells and leiomyosarcoma cells. The antitumor activity of eribulin in ovarian cancers has not been fully explored, so the present study aimed to determine the antitumor efficacy of eribulin and the involvement of stathmin in ovarian cancers. In a xenograft model of ovarian cancer, eribulin treatment reduced the tumor weight, which was accompanied by an increased level of phosphorylated stathmin. Eribulin stimulated the phosphorylation of stathmin in cultured cancer cell lines. The eribulin-induced phosphorylation of stathmin was inhibited by treatment with FTY720, an activator of protein phosphatase 2A (PP2A), and eribulin downregulated the expression of PP2A subunits. Furthermore, stathmin knockdown abrogated the inhibitory effects of eribulin on cell viability. Eribulin enhanced the antiproliferative effects of paclitaxel and concomitantly decreased stathmin expression. These results suggest that eribulin-induced phosphorylation of stathmin, mediated in part by PP2A downregulation, reduces stathmin activity and enhances the antiproliferative effects of paclitaxel in ovarian cancer. Collectively, the results of this study indicate that eribulin may suppress the proliferation of ovarian cancer cells partly by regulating the activity of stathmin.
Ovarian cancer has a poor prognosis and is one of the major causes of death in patients with gynecologic malignancies due to its late detection and the high level of angiogenesis.1,2) The introduction of antiangiogenic drugs such as bevacizumab, and poly(ADP-ribose) polymerases (PARP)-inhibitors such as olaparib or niraparib, has highlighted the potential of novel pharmacological treatments for ovarian cancers.2) However, standard treatments have remained largely unaltered over the past few decades, and platinum-based chemotherapy combined with paclitaxel is still the first-line therapy.3) Ovarian clear-cell carcinoma has a high risk of recurrence and is less sensitive to conventional platinum-based chemotherapy than other types of ovarian cancers.4) There is a need for novel effective agents for treating ovarian cancer.
Stathmin is a highly conserved phosphoprotein that plays an essential role in modulating microtubule dynamics by sequestering tubulin heterodimers and promoting microtubule catastrophe.5) Various kinases including protein kinase A (PKA) or protein phosphatases such as protein phosphatase 2A (PP2A) modulate the phosphorylation status of stathmin; phosphorylation of stathmin switches off its microtube depolymerization activity.6,7) The expression of stathmin is upregulated in a variety of human malignancies including ovarian cancer, and it is expressed in a stage-dependent manner in ovarian cancers.8,9) The microtubule-destabilizing activity of stathmin may be closely related to tumorigenesis and metastasis via promotion of epithelial to mesenchymal transition (EMT).10,11)
Eribulin is a nontaxane microtubule inhibitor that interrupts the tumor microenvironment.12) The mechanism of action of eribulin, which binds to the plus ends of microtubules, differs from that of other tubulin-binding agents such as vinca alkaloids and taxanes.13) Eribulin is approved for treating metastatic breast cancer and sarcomas.14) Eribulin has antitumor effects in pancreatic and lung cancer cells as well as ovarian cancer cells.15–17) Moreover, eribulin induces tumor vascular remodeling in human breast cancers and sarcomas, and reverses the EMT phenotype to a mesenchymal to epithelial transition (MET) phenotype in breast cancer.18,19) Furthermore, eribulin inhibits the binding of stathmin to tubulin.20) Of note, our recent studies showed that the antiproliferative activity of eribulin may be associated with the modulation of stathmin dynamics in breast cancer cells.21) Although the inhibitory effect of eribulin on ovarian cancer proliferation has been reported, the relationship between the efficacy of eribulin and stathmin dynamics has not been characterized in ovarian cancer.
The present study was therefore undertaken to examine the correlation between the antitumor effect of eribulin and stathmin dynamics in human epithelial ovarian cancer cells.
Eribulin was kindly provided by Eisai Co., Ltd. (Tokyo, Japan). H89 was purchased from the D. Western Therapeutics Institute, Inc. (Nagoya, Japan). KN-62 and FTY720 were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). The above reagents were dissolved in dimethyl sulfoxide (DMSO). Ovarian cancer cell lines, SK-OV-3 (serous carcinoma), and ES-2 cells (clear cell carcinoma) were obtained from the American Type Culture Collection (VA, U.S.A.), and RMG-1 cells (clear cell carcinoma) were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). RMG-1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM/F-12; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS). ES-2 cells were cultured in McCoy’s 5A modified medium from Thermo Fisher Scientific Inc. (Carlsbad, CA, U.S.A.) supplemented with 10% FBS. SK-OV-3 cells were maintained in RPMI1640 (FUJIFILM Wako Pure Chemical Corporation) containing 10% FBS.
Animals and Experimental DesignFemale BALB/cSlc-nu/nu nude mice (10 weeks old) were purchased from Japan SLC (Shizuoka, Japan). All procedures were reviewed and approved by the Institutional Animal Care Committees at Tokyo University of Pharmacy and Life Sciences (Approval No. P21-59). The mice were euthanized with isoflurane and subcutaneously implanted with RMG-1 cells (2 × 106 cells in 0.15 mL of Matrigel, Corning Inc., Corning, NY, U.S.A.) on both flanks. Two to three weeks after the implantation, when the tumors reached a mean volume of 550 mm3, the mice were randomly allocated into three groups: (i) vehicle control (n = 7), and (ii) eribulin-treated (1 mg/kg, n = 8) and (iii) eribulin-treated (3 mg/kg, n = 7) groups. The first day of the eribulin treatment was defined as day 0. Eribulin (1 or 3 mg/kg) was administrated via the tail vein. On day 4, animals received a second injection of eribulin (1 or 3 mg/kg). On day 8, mice were euthanized with isoflurane, and the tumors were separated, washed with phosphate-buffered saline (PBS), and weighed. About one-half of each tumor tissue was fixed in SUPER FIX KY500 (Kurabo Industries Ltd., Osaka, Japan) and embedded in paraffin, and then sections were prepared for immunohistochemistry. The remaining tissues were lysed for immunoblot analysis.
Immunohistochemical AnalysisParaffin-embedded sections of tumor tissues were stained with antibodies against phosphorylated (p)-stathmin, proliferating cell nuclear antigen (PCNA), and CD31 following a standard procedure.22) Briefly, paraffin sections were rehydrated, boiled for 20 min in 10 mM citrate buffer (pH 6.0), and then blocked with 10% normal goat serum for 1 h. The sections were incubated with an antibody against p-stathmin (p-stathmin Ser15; GWB-B00CF2, Genway Biotech, San Diego, CA, U.S.A., 1 : 200 dilution), PCNA (clone PC10, DAKO, Glostrup, Denmark, 1 : 200 dilution), or CD31 (D8V9E, Cell Signaling Technology, Beverly, MA, U.S.A., 1 : 200 dilution), and then incubated with Histofine simple stain MAX-PO (Nichirei Biosciences Inc., Tokyo, Japan). The sections were developed with 3,3′-diaminobenzidine solution (FUJIFILM Wako Pure Chemical Corporation) and counterstained with hematoxylin. The negative controls were incubated with rabbit immunoglobulin G (IgG) instead of a primary antibody.
Immunoblot AnalysisTumor tissue samples were homogenized in ice-cold radio immunoprecipitation assay (RIPA) lysis buffer (Cell Signaling Technology) and then centrifuged at 10000 × g for 10 min at 4 °C. Cell lysates were prepared in RIPA buffer. Equal amounts of protein (20 µg) were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, U.S.A.). The membranes were probed with primary antibodies specific for (p)-stathmin (GWB-B00CF2, 1 : 2500 dilution), stathmin (D1Y5A, Cell Signaling Technology, 1 : 2500 dilution), PP2Aa (81G5, Cell Signaling Technology, 1 : 2500 dilution), PP2Ab (100C1, 1 : 2500 dilution, Cell Signaling Technology), PP2Ac (52F8, 1 : 2500 dilution, Cell Signaling Technology), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; clone 5A12, FUJIFILM Wako Pure Chemical Corporation, 1 : 5000 dilution). Immunoreactive bands were detected using an enhanced chemiluminescence system (Western Lightning, PerkinElmer, Inc., Waltham, MA, U.S.A.) after incubation with horseradish peroxidase-labeled secondary antibodies (Vector Laboratories, Burlingame, CA, U.S.A.). The relative density of the bands was assessed by densitometry analysis of the digitalized autographic images using ImageJ software (NIH, Bethesda, MD, U.S.A.).
RNA Extraction and Real-Time RT-PCRTotal RNA was extracted using Isogen II (Nippon Gene, Tokyo, Japan). RNA (100 ng) was amplified by real-time RT-PCR using the SYBR Green Luna universal one-step RT-qPCR kit (New England Biolabs, Beverly, MA, U.S.A.). The primers used are listed in Table 1. The expression level of each mRNA was normalized to that of GAPDH as a reference transcript and analyzed using the comparative Ct method.22)
| Name (accession number) | Sequence | Product length (bp) |
|---|---|---|
| SET (NM_001122821.2) | F:5′-CATCTGAATGAGAGTGGTGATCC-3′ | 133 |
| R:5′-TCTCTGGTTCCTCATGCTGCCT-3′ | ||
| PPME1 (NM_016147.3) | F:5′-GGAAGTTCCAGATGCAGGTCCT-3′ | 161 |
| R:5′-AGCAGGTCACTAACAGCCAGGA-3′ | ||
| CIP2A (NM_020890.3) | F:5′-TGTGGCTCTACTGCGCTGGTTA-3′ | 126 |
| R:5′-TCAGCCGAGGAACAGTTAGCAG-3′ | ||
| STMN1 (NM_203401.2) | F:5′-TTCCCCTCCAAAGAAGAAGG-3′ | 94 |
| R:5′-GACCTCAGCTTCATGGGACT-3′ | ||
| PPP2R1A (NM_014225.6) | F:5′-ACCGCATGACTACGCTCTTCTG-3′ | 127 |
| R:5′-TTGAAGCGGACATTGGCAACCG-3′ | ||
| PPP2CA (NM_002715.4) | F:5′-GGTGGTCTCTCGCCATCTATAG-3′ | 109 |
| R:5′-CTGGATCTGACCACAGCAAGTC-3′ | ||
| GAPDH (NM_002046.7) | F:5′-AGCCACATCGCTCAGACA-3′ | 66 |
| R:5′-GCCCAATACGACCAAATCC-3′ |
Stathmin-silencing siRNA and nontargeting control siRNA (universal negative control) were purchased from Sigma-Aldrich. The sequences of stathmin siRNAs used were as follows: 5′-AUUGAGAUUCUUCUGCUCCUUGAGG-3′ and 5′-CCUCAAGGAGCAGAAGAAUCUCAAU-3. RMG-1, SK-OV-3, and ES-2 cells, grown to 50% confluency, were transfected with control siRNA (50 pmol/well) or stathmin siRNA (50 pmol/well) using Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, U.S.A.). After transfection for 24 h, cells were cultured in fresh medium for 24 h and then treated for 24 h with DMSO or eribulin.
Cell Viability AssayCell viability was assessed using a WST-8 assay (cell counting kit-8; Dojindo, Kumamoto, Japan). Briefly, cells were incubated with WST-8 reagent for 1 h, and the staining intensity of the medium was measured by absorbance at 460 nm. Data are expressed relative to the control value.
Statistical AnalysisData are expressed as the mean ± standard error of the mean (S.E.M.) for at least three independent experiments performed in duplicate. Statistical significance was determined using the Tukey–Kramer multiple comparisons test. A p-value of <0.05 was considered statistically significant.
To examine the antitumor effect of eribulin on ovarian cancer, we used a xenograft tumor model with subcutaneously implanted RMG-1 cells. The mice were randomly divided into three groups and treated with eribulin (1 or 3 mg/kg) according to the schedule shown in Fig. 1A. Administration of eribulin reduced the tumor weight in a dose-dependent manner (Figs. 1B, C). In agreement with the eribulin-mediated decrease in tumor weight, the staining intensity of PCNA, a marker of cell proliferation, was lower in eribulin-administrated tumors (Fig. 1D). Furthermore, tumor cells with an enlarged cell body and nuclei were observed in the eribulin-treated groups (Fig. 1D, arrows). Additionally, CD31-stained tumor vessels were smaller in size in eribulin-treated groups than in the control group (Fig. 1D).
A: The ovarian cancer xenograft model and the schedule of eribulin administration. RMG-1 cells were subcutaneously injected into the back of nude mice. Eribulin was intravenously administrated at a dose of 1 or 3 mg/kg eribulin on day 1 and day 4. Tumor samples were collected on day 8, and the weight of the tumor was measured. B: Representative photographs of tumors. Scale bar = 10 mm. C: Tumor weight. D, E: Immunohistochemical analysis of PCNA, CD31, and phosphorylated (p)-stathmin expression in the tumors. Scale bar = 100 µm. F: Immunoblot analysis of the expression of p-stathmin, stathmin, and the PP2A subunits PP2Aa/c. GAPDH was used as a loading control. G: Densitometry analysis of p-stathmin expression from the immunoblot analysis (F). Values represent the mean ± S.E.M. ** p < 0.01, *** p < 0.001 vs. control (ctrl).
To investigate whether eribulin alters stathmin dynamics in the xenograft model, we assessed the phosphorylation of stathmin in the tumor (Figs. 1E, F). Intensive staining of phosphorylated stathmin was observed in the eribulin-treated tumors, and the staining was prominent in the enlarged cells (Fig. 1E, arrowheads). Immunoblot analysis also revealed increased phosphorylation of stathmin in eribulin (3 mg/kg)-treated tumors (Figs. 1F, G).
As the phosphorylation status of stathmin is regulated by protein phosphatases including PP2A and protein kinases,6,7) we investigated first the effect of eribulin on PP2A expression (Fig. 1F). PP2A is a heterotrimeric holoenzyme consisting of a core dimer composed of scaffolding A subunit (PP2Aa) and a catalytic C subunit (PP2Ac), which associates with a regulatory B subunit (PP2Ab).23,24) Immunoblotting showed that eribulin treatment downregulated the expression of PP2Aa, but not PP2Ac (Fig. 1F), in the xenograft model.
Eribulin Inhibits the Proliferation of Ovarian Cancer Cell Lines and Modulates Stathmin DynamicsTo explore the antiproliferative effect of eribulin in ovarian clear cell carcinoma cells, RMG-1 and ES-2, and serous carcinoma SK-OV-3 cell lines were treated with different concentrations of eribulin (1, 10, or 100 nM) for 1 or 2 d (Fig. 2A). Eribulin treatment decreased cell viability in a concentration- and time-dependent manner in the cell lines. The effects of eribulin and the microtubule stabilizer paclitaxel on the phosphorylation and expression of stathmin were assessed. Eribulin concentration-dependently induced phosphorylation of stathmin and downregulated the level of stathmin protein in all cell lines (Figs. 2B, C). By contrast, paclitaxel had little effect on the level of stathmin phosphorylation in the cells (Figs. 2B, C). However, STMN1 mRNA expression was not altered in the cells (Fig. 2D).
A: Ovarian cancer cell lines (RMG-1, ES-2, and SK-OV-3 cells) were treated for 1 or 2 d with various concentrations of eribulin (1, 10, or 100 nM). The cell viability was evaluated by a WST-8 assay (n = 5). B: Cells were treated for 1 d with eribulin (2 or 10 nM) or paclitaxel (PTX, 10 nM). Cell lysates were subjected to immunoblot analysis to detect phosphorylated (p)-stathmin and total stathmin expression. GAPDH was used as a loading control. The representative results are shown. C: The relative expression level of target proteins (B), normalized to that of stathmin or GAPDH is shown (n = 3). D: Real-time RT-PCR analysis of STMN1 expression (n = 4). Values represent the mean ± S.E.M. from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DMSO.
To clarify the molecular mechanisms of eribulin-induced stathmin phosphorylation, the effects of the PKA inhibitor H89, the CaMKII inhibitor KN62, and the PP2A activator FTY720 on eribulin-mediated stathmin phosphorylation were examined (Fig. 3A, B). Pretreatment of cells with FTY720 repressed eribulin-induced stathmin phosphorylation, whereas H89 and KN62 had no significant effect, suggesting that PP2A is involved in stathmin phosphorylation in ovarian cancer cells. Eribulin reduced the protein level of stathmin, but the reduced expression was not affected by any inhibitors or activator, based on the densitometrical analyses (Fig. 3B, bottom). As we showed that PP2A expression was decreased in eribulin-treated tumors in the ovarian cancer xenograft model (Fig. 1F), we examined the effect of eribulin on PP2A expression in vitro (Fig. 3C). Eribulin treatment decreased the expression of PP2Aa in RMG-1 and SK-OV-3, but not ES-2. Eribulin downregulated the expression of PP2Ab and PP2Ac subunits in the three cell lines. On the contrary, eribulin did not alter the expression of PPP2R1A (PP2Aa) and PPP2CA (PP2Ac) mRNA (Fig. 3D). FTY720 has been reported to disrupt the interaction between PP2A and the endogenous PP2A inhibitor SET, leading to the activation of PP2A.25) We, therefore, examined the effect of eribulin on the endogenous PP2A inhibitory factors SET nuclear proto-oncogene (SET),25) protein phosphatase methylesterase 1 (PPME1),26) and cancerous inhibitor of protein phosphatase 2A (CIP2A).27) Eribulin treatment decreased slightly, but significantly the expression of SET in RMG-1 cells and increased the expression of CIP2A in SK-OV-3 cells, but did not affect SET, CIP2A, or PPME1 expression in the other cell lines tested (Fig. 3E).
A: Cells were treated for 1 h with CaMKII (KN62, 10 µM), PKA (H89, 10 µM) inhibitors, or a PP2A activator (FTY-720, 5 µM) and then stimulated with eribulin (10 nM) for 1 d. Cell lysates were subjected to immunoblot analysis to detect phosphorylated (p)-stathmin and stathmin expression. GAPDH was used as a loading control. The representative results are shown. B: The relative expression level of target proteins (A), normalized to that of stathmin or GAPDH is shown (n = 4). C: Cells were treated for 1 d with 10 nM eribulin. The expression of PP2A subunits (PP2Aa, PP2Ab, and PP2Ac) was determined using immunoblot analysis (n = 3). D: Real-time RT-PCR analysis of PPP2R1A and PPP2CA expression (n = 3). E: Real-time RT-PCR analysis of SET, CIP2A, and PPME1 expression (n = 3). Values represent the mean ± S.E.M. from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DMSO. # p < 0.05, ## p < 0.01 vs. Eribulin.
To explore the significance of stathmin expression in the antiproliferative activity of eribulin, RMG-1, ES-2, and SK-OV-3 cells were transfected with stathmin-targeting siRNA (Fig. 4). Immunoblotting confirmed that siRNA knockdown of stathmin abolished protein expression, and eribulin decreased stathmin protein expression (Fig. 4A). Similar to the results in Fig. 2A, eribulin treatment significantly decreased the viability of the control siRNA-transfected cells (Fig. 4B). Stathmin knockdown alone decreases cell viability. However, eribulin did not significantly reduced the viability of stathmin-silenced cells (DMSO vs. Eribulin).
Cells were transfected with nontargeting control (Ctrl si) or stathmin-targeting (stathmin si) siRNA for 2 d, and then stimulated with or without eribulin (10 nM) for 1 d. A: Immunoblot analysis of stathmin expression. GAPDH was used as a loading control. B: WST-8 assay analysis of cell viability (n = 4). Values represent the mean ± S.E.M. from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DMSO in Ctrl si.
Although paclitaxel is widely used for treating ovarian cancers, cancers that overexpress stathmin show low sensitivity to paclitaxel. As shown in Figs. 2 and 4, eribulin downregulated stathmin expression in ovarian cancer cells, and this observation led us to examine whether eribulin could modulate the antiproliferative activity of paclitaxel. RMG-1 cells were first cultured with or without eribulin and subsequently treated with either eribulin or paclitaxel (Fig. 5A). Twice treatments with eribulin significantly reduced the cell viability. Although a single treatment with paclitaxel (100, 250, or 500 nM) had little effect on the cell viability, the effect of paclitaxel was potentiated by pretreatment with eribulin (Fig. 5B). Once and twice treatment with eribulin significantly suppressed stathmin expression. Thus, stathmin expression was also markedly reduced by eribulin under the conditions different from those shown in Figs. 2 and 3. Paclitaxel alone (100, 250 nM) did not influence stathmin levels, although a high concentration of paclitaxel (500 nM) suppressed the expression (Figs. 5C, D). These results support the notion that the enhanced antiproliferative effect of the combination of eribulin and paclitaxel may be associated with the decreased expression of stathmin.
A: The schedule of the experiment. Cells were incubated for 1 d in the presence or absence of 10 nM eribulin (first treatment, 1st) and then additionally treated for 1 d with eribulin (10 nM) or paclitaxel (PTX, 100, 250, or 500 nM) (second treatment, 2nd). B: Cell viability was analyzed by a WST-8 assay (n = 4). Values represent the mean ± S.E.M. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Ctrl. * p < 0.05, *** p < 0.001 vs. PTX. C: The representative result of immunoblot analysis for stathmin expression. GAPDH was used as a loading control. D: The relative expression level of stathmin (C), normalized to that GAPDH is shown (n = 3). Values represent the mean ± S.E.M. * p < 0.05, ** p < 0.01 vs. no treatment.
The present study showed that eribulin suppressed the growth of ovarian tumor xenografts and increased stathmin phosphorylation. Decreased PCNA expression in eribulin-treated tumors suggests that eribulin has direct antiproliferative effects on tumor cells. In addition, eribulin abolished the formation of blood vessels with large lumens and may cause immature vascularization in the tumor tissues. Angiogenesis is indispensable for the establishment and proliferation of tumors. Consistent with our observation, several recent studies have demonstrated that eribulin induces remodeling of the tumor vasculature in a mouse xenograft model of breast cancer and in patients with breast cancer.18,28,29) Therefore, our results suggest that eribulin suppresses ovarian cancer tumorigenesis by abrogating tumor vascularization. Our previous reports show that stathmin knockdown downregulates the angiogenesis-associated factors hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) by repressing phosphatidylinositol 3-kinase (PI3k)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling in an ovarian cancer cell line and human umbilical vein endothelial cells; these findings also suggest a role for stathmin in the development of ovarian cancer.30,31) Thus, eribulin may exert antitumor activity through a direct action on cancer cells as well as affect the growth of ovarian tumor vessels.
We observed that eribulin treatment increased the phosphorylation of stathmin by downregulating the protein level of PP2A subunits in ovarian cancer cells. Of note, neither the mRNA expression of PP2A subunits nor the expression of endogenous PP2A inhibitory factors were altered by eribulin treatment in vitro, except SET in RMG-1 cells and CIP2A in SK-OV-3 cells. These results imply that eribulin induces stathmin phosphorylation by downregulating PP2A expression without altering expression of its inhibitory factors in ovarian cancer cells. The proapoptotic protein Siva1 is overexpressed in ovarian cancer cell lines, and enforced expression of Siva 1 inhibits proliferation, promotes apoptosis, and represses migration and invasion by facilitating phosphorylation of stathmin in these cells.32) Thus, eribulin-mediated stathmin phosphorylation may contribute to the antiproliferative effects of eribulin.
Our previous studies demonstrated that eribulin caused phosphorylation of stathmin by not only downregulating PP2A levels but also by activating PKA and CaMKII pathways in breast cancer cells.21) By contrast, eribulin-induced phosphorylation of stathmin was not affected by PKA and CaMKII inhibitors in ovarian cancer cells. In addition, paclitaxel enhances stathmin phosphorylation in acute lymphoblastic leukemia cells and increases PP2A expression in non-small cell lung cancer (NSCLC) cells.33,34) However, paclitaxel had no effect on stathmin phosphorylation and PP2A expression in ovarian cancer cells in this study. These results suggest that phosphorylation of stathmin induced by microtubule-associated drugs, including eribulin and paclitaxel, might be differently regulated in different types of cancer cells.
Stathmin is highly expressed in various cancers including ovarian cancers, and overexpression of stathmin is an indicator of poor prognosis in ovarian cancer.35) In the present study, we showed that eribulin attenuated stathmin protein levels, but not mRNA expression, in ovarian cancer cells. Although the precise mechanisms of eribulin-induced stathmin downregulation are unclear, eribulin may post-transcriptionally modulate stathmin protein levels in ovarian cancer cells. It is conceivable that eribulin-mediated downregulation of stathmin levels may also in part contribute to the antiproliferative effect of eribulin. Of note, the present study showed that knockdown of stathmin expression suppressed the antiproliferative effect of eribulin in ovarian cancer cells. This is in line with our recent findings showing that stathmin knockdown abrogates the inhibitory effect of eribulin on cell viability in breast cancer cell lines.21) Furthermore, overexpression of stathmin increases the antiproliferative effect of eribulin in breast cancer cells.21) These results indicate that stathmin is likely involved in the antiproliferative effect of eribulin and suggest that eribulin can effectively exert its action in ovarian cancer cells overexpressing stathmin.
Combined therapy with paclitaxel and carboplatin is the first-line chemotherapy for ovarian cancers; however, a large number of patients develop drug resistance.4) The overexpression of stathmin causes resistance to paclitaxel treatment in lung, breast, and endometrial cancer; these findings indicate that paclitaxel sensitivity presumably depends on the level of stathmin expression.36–38) Additionally, the combination of paclitaxel treatment and stathmin knockdown inhibits tumor growth and enhances apoptosis in nasopharyngeal and esophageal carcinoma.34,39) In the current study, the combination of eribulin and paclitaxel enhanced the antiproliferative effect of paclitaxel in RMG-1 cells. Eribulin treatment decreased stathmin expression in paclitaxel-treated cells. Taken together, these results suggest that eribulin might improve the therapeutic efficacy of paclitaxel against drug-resistant cancer cells by downregulating the expression of stathmin. Among the various types of ovarian cancers, clear-cell carcinoma has an especially poor response to chemotherapy,40) so there is a need to develop novel drugs or other therapeutics. In the current study, eribulin potentiated the effect of paclitaxel in the ovarian clear-cell carcinoma cell line RMG-1. This finding implies that combination therapy with eribulin and subsequent paclitaxel could be useful for treating ovarian clear-cell carcinoma. Further studies regarding the molecular mechanisms by which eribulin regulates stathmin phosphorylation and abundance are required to support our findings.
In summary, our results suggest that eribulin-triggered stathmin phosphorylation and downregulation could be mechanisms that mediate the antitumor effect of eribulin on ovarian cancer cells. Our findings show new molecular mechanisms of action of eribulin in ovarian cancer cells. In addition, we propose that combined treatment with eribulin and paclitaxel could be effective in ovarian cancer.
We are grateful to Eisai Co., Ltd. for providing eribulin.
The authors declare no conflict of interest.
