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. 2015 Jun 20;6(17):15194-208.
doi: 10.18632/oncotarget.3827.

Elevated MARCKS phosphorylation contributes to unresponsiveness of breast cancer to paclitaxel treatment

Ching-Hsien Chen  1 , Chun-Ting Cheng  2   3 , Yuan Yuan  4 , Jing Zhai  5 , Muhammad Arif  1 , Lon Wolf R Fong  1 , Reen Wu  1 , David K Ann  2   3
Affiliations

Elevated MARCKS phosphorylation contributes to unresponsiveness of breast cancer to paclitaxel treatment

Ching-Hsien Chen et al. Oncotarget. .

Abstract

Accumulating evidence has suggested that myristoylated alanine-rich C-kinase substrate (MARCKS) is critical for regulating multiple pathophysiological processes. However, the molecular mechanism underlying increased phosphorylation of MARCKS at Ser159/163 (phospho-MARCKS) and its functional consequence in neoplastic disease remain to be established. Herein, we investigated how phospho-MARCKS is regulated in breast carcinoma, and its role in the context of chemotherapy. In a screen of patients with breast tumors, we find that the abundance of phospho-MARCKS, not MARCKS protein per se, increased in breast cancers and positively correlated with tumor grade and metastatic status. Among chemotherapeutic agents, mitotic inhibitors, including paclitaxel, vincristine or eribulin, notably promoted phospho-MARCKS accumulation in multiple breast cancer cells. We further show that phospho-MARCKS acted upstream of Src activation upon paclitaxel exposure. Reduction of phospho-MARCKS by knockdown of MARCKS or pharmacological agents increased paclitaxel sensitivity. Particularly, a known phospho-MARCKS inhibitor, MANS peptide, was demonstrated to increase paclitaxel efficacy and attenuate angiogenesis/metastasis of xenografted breast cancer cells by decreasing abundance of phospho-MARCKS and messages of inflammatory mediators. Our data suggest that unresponsiveness of breast cancer to paclitaxel treatment is, at least in part, mediated by phospho-MARCKS and also provide an alternative therapeutic strategy against breast cancer by improving taxanes sensitivity.

Keywords: MANS peptide; breast cancer; mitotic inhibitor; paclitaxel; phospho-MARCKS.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that they have no competing financial interests with the study.

Figures

Figure 1
Figure 1. Elevated phospho-MARCKS abundance in invasive breast cancer
A. Correlation of high phospho-MARCKS levels with distant metastasis of breast cancer. Left, representative images of immunohistochemical (IHC) staining in adjacent non-tumor areas and breast cancer specimens with low levels of phospho-MARCKS (score = +1 or +2) and high phospho-MARCKS abundance (score = +3). Right, percentage of patients with high and low phospho-MARCKS according to no distant metastasis (M0) vs distant metastasis (M1). P = 0.042, Fisher's exact test. B. Left, representative IHC images of breast primary tumors and lymph node metastatic tumors by using anti-phospho-MARCKS and anti-MARCKS antibodies. Right, percentage of patients with high and low phospho-MARCKS according to cancer types (noninvasive ductal carcinomas in situ, invasive breast carcinomas and lymph node metastatic tumors). p = 0.048, Fisher's exact test. (C-D) Higher phospho-MARCKS promotes breast cancer cell invasion and migration. MDA-MB-231 cells were transduced with control non-specific or MARCKS-specific shRNA-containing lentiviruses. Following knockdown of MARCKS, cells were re-expressed either wild type or mutant (S159/163A) V5-tagged MARCKS. C. Left, the levels of phospho-MARCKS abundance and MARCKS expression in these genetically modified cells were determined by Western blot. These cells were plated on Transwells with matrigel; 20 hours later, migrated cells were fixed, stained, and counted using light microscopy. A representative picture of each group is shown in the middle. Right, quantification of migrated cells to the lower chamber. Data expressed as mean ± SD (n = 4), *p < 0.05 as compared to cells receiving control shRNA. D. Confluent cultures of these cells were scratched and wound healing repair was monitored microscopically after the scratch. Left, representative phase contrast pictures. Right, numbers of cells migrated to the wound area were quantified at 16 hours post-scratching. (n = 4, *p < 0.05 versus control shRNA).
Figure 2
Figure 2. MARCKS inhibition is associated with increased paclitaxel cytotoxicity
A. Analysis of phospho-MARCKS levels in breast cancer cells after exposure to chemotherapy. Cells were incubated with various chemotherapeutic agents including paclitaxel (20 nM), cisplatin (15 μM), doxorubicin (25 nM) and etoposide (5 μM), respectively. After 24 hours, cells were collected and subjected to Western blot analysis. B-C. Knockdown of MARCKS to down-regulate phospho-MARCKS levels decreases cell viability of MDA-MB-231 B. and MDA-MB-468 C. cells in response to chemotherapy. Cells were transduced with control non-specific or MARCKS-specific shRNA-containing lentiviruses. These cells were subjected to 20 nM paclitaxel, 15 μM cisplatin, 25 nM doxorubicin or 5 μM etoposide for treatment. After 72 hours, cell viability was assessed by Trypan blue staining (left). Data shown as mean ± SD; *: p < 0.05 versus control shRNA (n = 4). Right, Western blot analysis of cleaved caspase-3 and PARP in control shRNA and MARCKS-knockdown cells after 48 hours of treatment with 20 nM paclitaxel.
Figure 3
Figure 3. Phospho-MARCKS increases in response to mitotic inhibitors treatment
A. Paclitaxel treatment induces activation of MARCKS and Src in TNBC cells. Cells were incubated with various doses of paclitaxel as indicated. After 24 hours, cells were collected and subjected to Western blot analysis. B-C. Determination of the major kinase that led to MARCKS activation in these TNBC cells in response to paclitaxel. B, cells were co-treated with paclitaxel (10 nM) and PKC inhibitor (Calphostin C; 250 nM). C, cells were co-treated with paclitaxel (10 nM) and Src inhibitor (Dasatinib; 100 nM). After 24 hours of co-treatment, Western blots were carried out with specific antibodies as indicated. D. Western blot analysis of phospho-MARCKS levels in MDA-MB-468 cells upon treatment with various mitotic inhibitors for 24 hours. E. Eribulin-treated cells were incubated with or without PKC inhibitor (Ro 31-8220) for 24 hours and then subjected to immunoblotting analysis.
Figure 4
Figure 4. Co-treatment of MARCKS inhibitor and paclitaxel suppresses proliferation of breast cancer
A. MARCKS inhibitor MANS peptide inhibited paclitaxel-induced MARCKS phosphorylation of breast cancer cells. MDA-MB-231 (left) and MDA-MB-468 (right) cells were pre-treated with 100 μM MANS peptide for 12 hours and then co-treated with paclitaxel or left alone as indicated. These cells were harvested 24 hours later and subjected to immunoblot analysis. (B-C) The combinatorial effect of MANS peptide with paclitaxel on breast cancer cell lines. MDA-MB-231 B. and MDA-MB-468 C., were co-treated with various dosages of paclitaxel and 100 μM MANS peptide. After 72 hours, cell viability was determined by MTS assay. *: p < 0.05. Right table, estimated IC50 values for paclitaxel alone and combined treatments with MANS peptide. D. Cells were treated with the indicated concentrations of MANS peptide combined with paclitaxel and colonies were counted after 10 days by using crystal violet staining. Left, data are representative of three independent experiments. Right, expression of colony formation index. E. Caspase-3 activation in MANS-treated cells upon paclitaxel treatment. Cells were incubated in a medium containing either paclitaxel, MANS peptide or combination of paclitaxel and MANS peptide for 48 hours and then subjected to Western blot analyses.
Figure 5
Figure 5. Targeting phospho-MARCKS improves paclitaxel efficiency in vivo
A-B. Growth curves A. and tumor weights B. of xenograft tumors generated by orthotopic injection of MDA-MB-468 cells into the fat pads of mice breasts. Once tumor volume reached an average of 100 mm3 at the injected site, mice were randomly grouped for intraperitoneal injection, once every three days with either vehicle, MANS peptide (12.5 mg/kg), paclitaxel (3 mg/kg) alone or paclitaxel with MANS peptide (12.5 mg/kg). Tumor measurements were taken every three days after each drug injection and data were presented as mean ± SD (A). After 21 days of treatment, the xenograft tumors of these mice were removed and weighed. Data were expressed as the mean ± SE (B). *: p < 0.05 for paclitaxel + MANS as compared to paclitaxel alone (n = 5). C. H&E and immunohistochemical staining of phospho-MARCKS (Ser159/163), phospho-Src (Tyr416), PCNA and activated caspase-3 in xenograft tumors (n = 5) as described in B. Representative images are shown and positive staining is quantified (mean ± SD, *p < 0.05 versus vehicle group). T: tumor mass.
Figure 6
Figure 6. Suppression of phospho-MARCKS reduces microvessel density and the expression of angiogenic factors in TNBC cells
A-B. MDA-MB-468 cells were injected orthotopically into female nude mice as described in Figure 5. Tissues were stained with the endothelial marker CD31. Representative pictures are shown. A, CD31-stained cells in the most vascular areas (hot spots); microvessel density (MVD) is expressed as the percentage of CD31+ area per high-powered field. Analysis was performed on 6 to 12 fields with higher magnification per tumor with ImageJ software. Data shown as mean ± SD, *: p < 0.05 as compared to paclitaxel-treated group (top). Middle, CD31-stained cells in the intra-tumor area. Bottom, CD31-stained cells in the periphery of tumor mass by 40X and 200X (an enlarged image from the frame) magnification. A dark dotted line represents the boundary between tumor mass and adjacent tissue. B, cancer cells within the neovasculature of vehicle- and paclitaxel-treated tumors. C. MDA-MB-468 cells were transduced with MARCKS-specific or non-specific shRNA-containing lentiviruses. After 72 hours of transduction, expression of angiogenic factors as indicated were determined by quantitative RT-PCR (n = 3, *: p < 0.05 versus control shRNA). D. Down-regulation of angiogenic factors in MANS-treated cells. MDA-MB-468 cells were pre-treated with 100 μM MANS peptide for 12 hours and then co-treated with or without 10nM paclitaxel. After 24 hours of co-treatment, these cells were collected and subjected to quantitative RT-PCR analyses (n = 3, *: p < 0.05 versus paclitaxel-treated group).
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