- Email: [email protected]
radiotherapy response in vivo
Journal Pre-proof CD44 variant 6 is associated with prostate cancer growth and chemo-/radiotherapy response in vivo Jie Ni, Belamy B. Cheung, Julia Beretov, Wei Duan, Joseph Bucci, David Malouf, Peter Graham, Yong Li PII:
S0014-4827(20)30044-6
DOI:
https://doi.org/10.1016/j.yexcr.2020.111850
Reference:
YEXCR 111850
To appear in:
Experimental Cell Research
Received Date: 4 October 2019 Revised Date:
8 January 2020
Accepted Date: 13 January 2020
Please cite this article as: J. Ni, B.B. Cheung, J. Beretov, W. Duan, J. Bucci, D. Malouf, P. Graham, Y. Li, CD44 variant 6 is associated with prostate cancer growth and chemo-/radiotherapy response in vivo, Experimental Cell Research (2020), doi: https://doi.org/10.1016/j.yexcr.2020.111850. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
CRediT author statement Jie Ni: Conceptualization, Methodology, Investigation, Formal analysis, Writing - Original Draft, Visualization. Belamy Cheung: Conceptualization, Methodology, Resources. Julia Beretov: Investigation, Formal analysis. Wei Duan: Conceptualization, Methodology. Joseph Bucci: Resources, Writing - Review & Editing. David Malouf: Resources, Writing - Review & Editing. Peter Graham: Resources, Supervision, Writing - Review & Editing, Funding acquisition. Yong Li: Conceptualization, Supervision, Writing - Review & Editing, Funding acquisition.
CD44 variant 6 is associated with prostate cancer growth and chemo/radiotherapy response in vivo Jie Nia,b, Belamy B. Cheungc,d, Julia Beretova,b,e, Wei Duanf, Joseph Buccia,b, David Maloufa,g, Peter Grahama,b, Yong Lia,b,h,*
a
Cancer Care Centre, St George Hospital, Kogarah, NSW 2217, Australia
b
St George and Sutherland Clinical School, Faculty of Medicine, UNSW Sydney, NSW 2052,
Australia c
Children's Cancer Institute Australia, Lowy Cancer Research Centre, UNSW Sydney, NSW
2052, Australia d
School of Women's & Children's Health, Faculty of Medicine, UNSW Sydney, NSW 2052,
Australia e
Anatomical Pathology, NSW Health Pathology, St George Hospital, Kogarah, NSW 2217,
Australia f
School of Medicine and Centre for Molecular and Medical Research, Deakin University,
Waurn Ponds, VIC 3216, Australia g
Department of Urology, St George Hospital, Kogarah NSW 2217, Australia
h
School of Basic Medical Sciences, Zhengzhou University, Henan 450001, China
*Correspondence Yong Li, Cancer Care Centre, St George Hospital, Level 2, 4-10 South St, Kogarah NSW 2217 Australia. Tel: +61-2-91131338; Fax: +61-2-91131386; Email: [email protected]
1
ABSTRACT We have previously demonstrated that CD44 variant 6 (CD44v6) is associated with prostate cancer (CaP) growth and therapeutic resistance in vitro, however, the role of CD44v6 in CaP in vivo is not fully understood. The purpose of this study is to investigate the effect of CD44v6 on CaP growth and chemo−/radiotherapy response in NOD/SCID mouse models in vivo and to validate its role as a therapeutic target for CaP therapy. CD44v6 was knocked down in PC-3M CaP cell line using short hairpin RNA. Subcutaneous (s.c.) and orthotopic CaP mouse xenografts were established. The effect of CD44v6 knockdown (KD) on tumour growth was evaluated on both s.c. and orthotopic models. Chemo−/radiotherapy response was evaluated on the s.c. model. Association of CD44v6 with PI3K/Akt pathway was validated using immunohistochemistry staining. We found that KD of CD44v6 significantly reduced tumour growth in both models, and enhanced the sensitivity of tumours to chemotherapy and radiotherapy in the s.c. model. In addition, we demonstrated that KD of CD44v6 is associated with downregulation of the PI3K/Akt/mTOR pathway. Our data confirm that CaP growth and chemo−/radiosensitivity in vivo is associated with CD44v6, which holds great promises as a therapeutic target in the treatment of CaP.
Keywords CD44v6, prostate cancer, PI3K/Akt/mTOR, chemotherapy, radiotherapy
2
1. Introduction Prostate cancer (CaP) is the most prevalent cancer diagnosed in men in the western world, constituting nearly 20% of new cancer cases in the United States in 2018 [1]. One of the biggest therapeutic challenges of CaP is the development of resistance to radiation and chemotherapy. Radiation therapy, either brachytherapy or external beam radiation therapy, remains a mainstay in the treatment of CaP, particularly for the organ-confined disease. However, a small subset of cancer cells will acquire resistance during fractionated irradiation, and thereby survived the radiation, which leads to locoregional recurrence and distant metastasis [2]. Radioresistance is one of the major factors that limit the efficacy of radiotherapy, and radiosensitisation of cancer cells is of great significance for the CaP radiotherapy. Chemotherapy is the main treatment option for metastatic CaP. Despite extensive research on novel chemotherapy agents for over a decade, docetaxel (DTX) has been used as the firstline agents since 2004, following two Phase III trials [3, 4] where docetaxel demonstrated overall survival benefits in metastatic castration-resistant prostate cancer (mCRPC) patients. However, approximately half of mCRPC patients do not respond, and those who do will eventually develop resistance to docetaxel, posing a significant challenge in the treatment of late-stage CaP. CD44 variant 6 (CD44v6) is one of the isoforms of CD44 by alternative mRNA splicing and has been found to be closely associated with tumour behaviour in various cancers [5-7]. In particular, our previous study demonstrated that CD44v6 is associated with CaP proliferation, invasion and chemo-/radio-resistance in vitro [8]. However, few studies on the roles of CD44v6 in CaP chemo−/radioresistance in vivo are available. To address this, in the current study, we used subcutaneous (s.c.) and orthotopic CaP NOD/SCID mouse models to investigate the effect of CD44v6 on CaP growth and used the s.c. model to test the
3
chemo−/radiotherapy response. We demonstrate for the first time that CD44v6 is involved in tumour growth and chemo−/radiotherapy response in CaP animal models in vivo, and associated with the PI3K/Akt/mTOR pathway activation.
2. Methods 2.1. Antibodies and reagents Detailed information and conditions of antibodies used in the study are listed in Table S1. DTX was purchased from Hospira (Melbourne, VIC, Australia), and diluted in 0.9% saline for in vitro study. All cell culture reagents were purchased from Life Technologies Australia Pty Ltd (Melbourne, VIC, Australia) unless otherwise stated. MISSION® lentiviral transduction particles encoding for short hairpin RNA (shRNA) against CD44v6 and a nontarget shRNA control (scr) transduction particles were designed and obtained from SigmaAldrich Pty Ltd (Castle Hill, NSW, Australia). 2.2. Cell line and cell culture Androgen-non-responsive PC-3M CaP cell line was obtained from MD Anderson Cancer Center under Material Transfer Agreement and cultured in RPMI-1640 supplemented with 10% (vol/vol) FBS, 50 U/mL of penicillin, and 50 µg/mL of streptomycin. The cells were maintained in a humidified incubator at 37 °C and 5% CO2. The identity of PC-3M was confirmed by short tandem repeat profiling (CellBank Australia, NSW, Australia) and the assays were performed within a few passages of initial authentication. The cell line was regularly tested to confirm the absence of mycoplasma contamination using the LookOut® mycoplasma qPCR detection kit (Sigma-Aldrich Pty Ltd, NSW, Australia). 2.3. Short hairpin RNA (shRNA) transfection for CD44v6 PC-3M cells were seeded in 6-well plates, and upon reaching 70–80% confluence, the cells were infected with lentiviral particles lentivirus expressing CD44v6 shRNA or a scrambled 4
sequence. After 48 hours, the stable transduced cells were selected in puromycin-containing medium (500 µg/mL), propagated and finally maintained in medium containing 1 mg/mL puromycin for the following experiments. The KD were performed with three different custom-designed shRNAs that target non-overlapping regions of CD44v6 on PC-3M cell line. Detailed information on CD44v6-shRNAs is included in Table S2. 2.4. Western blot Briefly, 20 ug of extracted proteins were separated on Bis-Tris gels (Life Technologies Pty Ltd, Australia) and blotted onto PVDF membranes (Millipore, USA). The membrane was blocked with 5% bovine serum albumin in Tris-buffered saline with 0.1% Tween 20 and incubated overnight at 4 °C with the indicated primary antibodies (Table S1). Next, the membranes were washed and incubated with goat anti-rabbit/mouse IgG secondary antibody for 1 hour at room temperature. Protein expression was analysed using Clarity Max Western ECL Substrate (Bio-rad Laboratories, Australia) in the ImageQuant LAS4000 system (GE Healthcare, USA). Mouse anti-human β-tubulin monoclonal antibody was used as a loading control. 2.5. Quantitative real-time PCR (qRT-PCR) Total RNA was isolated using the High Pure RNA Isolation Kit (Roche Life Science, NSW, Australia) and 2 µg of total RNA was used to synthesise cDNA using the iScript™ cDNA Synthesis Kit (Bio-rad Laboratories, Australia). qRT-PCR was carried out on the CFX96™ Real-Time PCR Detection System (Bio-rad Laboratories, Australia) in a solution containing cDNA, primer and SsoAdvanced Universal SYBR® Green Supermix (Bio-rad Laboratories, Australia). GAPDH was used as a reference control. 2.6. Establishment of animal models All animal procedures in the study followed the protocols approved by the Animal Care and Ethics Committee (ACEC 14/46A) of UNSW Sydney, Australia.
5
Male, 6–8 weeks old NOD/SCID mice (Animal Resources Centre, WA, Australia) were housed in a group of 3 or 4, in specific pathogen-free facilities and all experiments were performed in a laminar flow cabinet. Mice were kept at least 1 week before experimental manipulation. All mice were monitored daily for health status and the weight was recorded at least twice a week. All mice remained healthy and active during the experiments. For the s.c. xenograft model, 2 × 106 PC-3M-CD44v6-KD or PC-3M-CD44v6-scr CaP cells in 100 µL phosphate-buffered saline (PBS) were implanted subcutaneously in the right rear flank region of mice. Tumour progression was recorded weekly by measurements using a calliper, and tumour volumes were calculated as follows: length×width×height×0.52 (in millimetres) for up to 8 weeks. For the orthotopic xenograft model, 1 × 106 PC-3M-CD44v6-KD or PC-3M-CD44v6-scr CaP cells suspended in 50 µL PBS were injected into the prostatic lobe followed by a lower midline laparotomy as previously described [9]. Starting from the second week after cell inoculation, tumour progression was monitored weekly by 3D-ultrasonography [10]. 2.7. In vivo chemosensitivity assay In this study, we used the s.c. mouse model to evaluate the effect of CD44v6 KD on tumour’s response to docetaxel. The dosage of docetaxel was determined by a maximum tolerated dose (MTD) assay set out in our previous study [9]. When the average tumour size reached 70±10 mm3 in each subgroup, 4 mice/per group (n=4) including PC-3M-CD44v6KD and PC-3M-CD44v6-scr groups were treated with 50 mg/kg DTX once by intraperitoneal injection, and 4 mice/per group (n=4) including PC-3M-CD44v6-KD and PC-3M-CD44v6scr groups were treated with saline as the vehicle control (VC). Tumour growth was calculated by measurements using a calliper. 2.8. In vivo radiosensitivity assay
6
We used s.c. mouse model to evaluate the effect of CD44v6 KD on tumour’s response to radiation. The radiation dosage was determined by a previous MTD assay [9]. When the tumour volume reached 70±10 mm3 in s.c. model, mice were anaesthetised with ketamine and xylazine, and then immobilised in a custom-designed chamber with a lead cover. Mice were placed in the X-RAD 320 Biological Irradiator (Precision X-Ray, North Branford, CT, USA) and irradiated with only the tumour area exposed. In each subgroup, 4 mice (n=4) were irradiated at a dose of 2Gy for 4 consecutive days, while 4 mice (n=4) went through the same procedure with the no irradiation as a sham control (SC). 2.9. Immunohistochemistry (IHC) IHC was performed on biological triplicate. Paraffin sections were de-paraffinised and rehydrated, followed by antigen retrieval in DAKO Low pH Target Retrieval Solution (Agilent, VIC, Australia) and then incubated with primary antibodies overnight at 4 °C. For CD31 staining, cryosections were fixed with ice-cold acetone for 10 min, and rinsed in Trisbuffered saline (TBS), before incubated with the CD31 primary antibody. After washing with TBS, slides were then incubated with HRP-conjugated secondary antibody (1:150 dilution) for 45 min at room temperature. Immunoreactivity was then developed with the DAKO Liquid DAB+ Kit (Agilent, VIC, Australia). Slides were then counterstained with Harris Haematoxylin (Thermo Fisher Pty Ltd, VIC, Australia) for 1 minute, and blued with Scott’s Bluing solution (Sigma-Aldrich Pty Ltd, NSW, Australia). Tissue sections were washed in water, dehydrated, cleared and mounted. 2.10. Assessment of immunostaining IHC intensity was assessed using a light microscope (Leica, Germany). The percentage of the tumour cells with positive staining was assessed. Staining intensity was graded between 0-3, following the criteria as follows: - (negative, 0–10%); 1+ (weak, 10–45%); 2+ (moderate,
7
45–70%); 3+ (strong, >70%) of the tumour tissue stained. The evaluation of staining intensity was done independently by two experienced observers (JN and JBeretov). 2.11. Statistical analysis All numerical data were expressed as the average of the values obtained, and the SD was calculated (mean ± SD). Data from different groups were compared using the two-tail student’s t-test. P<0.05 was considered significant. All numerical statistical analyses were performed using the GraphPad Prism 7 (GraphPad, CA, USA).
3. Results 3.1. Expression of CD44v6 in CD44v6-KD and control cells CD44v6 KD was confirmed using Western Blot and qRT-PCR. The KD were performed with three different custom-designed shRNAs that target non-overlapping regions of CD44v6, and after validation, CD44v6-shRNA#1 was selected for the following studies (Fig. S1). As shown in Fig. 1, following shRNA transfection, a reduction of CD44v6 expression on PC3M-CD44v6-KD cells were observed, compared to PC-3M-CD44v6-scr and PC-3M cell lines. No significant difference in the expression of CD44v6 was noted between PC-3MCD44v6-scr and PC-3M cell lines. 3.2. KD of CD44v6 downregulated tumour growth in both s.c. and orthotopic xenografts Weekly measurements of tumour size were plotted. As shown in Fig. 2, both s.c. and orthotopic PC-3M-CD44v6-KD xenografts showed a significant decreased tumour growing pattern compared to the PC-3M-CD44v6-scr group (n=9; P<0.05), respectively. In the s.c. model, all PC-3M-CD44v6-scr xenograft tumours became palpable from the second week post cell inoculation while all tumours from PC-3M-CD44v6-KD group became palpable from the third week after cell inoculation. Significant tumour growth difference was found 3 weeks post cell inoculation (Mean±SD: KD, 15.4±3.9 mm3; scr, 38.0±12.0 mm3; 8
P<0.05). At the end of the experiment (7 weeks after cell inoculation), tumours were harvested, measured and weighed. In PC-3M-CD44v6-KD
group, tumour volume was
260.6±21.5 mm3, tumour weight was 0.6±0.2g; whereas in PC-3M-CD44v6-scr group, tumour volume was 566.1±56.7 mm3, tumour weight was 1.9±0.2g (Mean±SD, P<0.001 in both measurements). In the orthotopic model, the prostatic tumour became visible from the second week post cell inoculation in both PC-3M-CD44v6-KD and PC-3M-CD44v6-scr groups. Significant tumour growth difference was found 3 weeks post cell inoculation (Mean±SD: KD, 42.9±3.3 mm3; scr, 58.9±2.6mm3; P<0.01). After 5 weeks post cell inoculation, in PC-3M-CD44v6KD group, tumour volume was 100.9±12.0 mm3, while in PC-3M-CD44v6-scr group, tumour volume was 231.2±26.0 mm3 (Mean±SD, P<0.01). At the end of the experiment (7 weeks after cell inoculation), tumours were harvested, measured and weighed. In PC-3M-CD44v6KD group, tumour volume was 254.6±37.7 mm3, tumour weight was 0.5±0.2g; whereas in PC-3M-CD44v6-scr group, tumour volume was 231.2±16.0 mm3, tumour weight was 1.7±0.2g (Mean± SD, P<0.01 in both measurements). 3.3. KD of CD44v6 affects CaP chemo-/radio-sensitivity in the s.c. xenograft model DTX or VC treatment was administered at 4 weeks post cell inoculation when the average tumour size reached 70±10 mm3. Tumour volumes were plotted against elapsed time (Fig. 3a). Significant differences were seen between PC-3M-CD44v6-KD-DTX and PC-3MCD44v6-KD-VC groups (n=4, P<0.01), PC-3M-CD44v6-scr-DTX and PC-3M-CD44v6-scrVC groups (n=4, P<0.01), and PC-3M-CD44v6-scr-DTX and PC-3M-CD44v6-scr-VC groups (n=4, P<0.05), respectively. The ratios of DTX-treated versus VC-treated tumour volumes (DTX/VC) were calculated and plotted from the start of DTX treatment to the end of experiments (Fig. 3b). Two groups responded to DTX treatment as early as 1 week posttreatment. Significant differences between two groups in response to DTX were first
9
observed 1 week post DTX treatment. Overall, the ratio of PC-3M-CD44v6-KD tumour volumes decreased faster in response to DTX, compared to the PC-3M-CD44v6-scr group (P<0.01). Notably, in PC-3M-CD44v6-KD, tumours on 75% (3 out of 4) mice became completely impalpable at the end of the experiment, while all PC-3M-CD44v6-scr tumours are still palpable, suggesting that the KD of CD44v6 can greatly increase the DTX sensitivity of CaP tumours in the s.c. mouse model. Fractionated RT (2Gy x 4 fractions) or sham radiation was given 4-week post cell inoculation when the average tumour size reached 70±10 mm3. Tumour volumes were plotted against elapsed time (Fig. 3c). Significant differences were seen between PC-3M-CD44v6KD-RT and PC-3M-CD44v6-KD-SC groups (n=4, P<0.01), PC-3M-CD44v6-scr-RT and PC-3M-CD44v6-scr-SC groups (n=4, P<0.01),
PC-3M-CD44v6-scr-SC and PC-3M-
CD44v6-scr-SC groups (n=4, P<0.05), respectively. The ratios of RT versus SC tumour volumes (RT/SC) were calculated and plotted from the start of RT to the end of experiments (Fig. 3d). PC-3M xenografts responded to RT from 1 week post-treatment. However, more significant differences between the two groups in response to RT were observed 3 weeks post-RT. Overall, the ratio of PC-3M-CD44v6-KD tumour volumes decreased faster in response to RT compared to the sham irradiation (P<0.05), suggesting that the KD of CD44v6 can increase the radiosensitivity of CaP tumours in the s.c. mouse model. 3.4. KD of CD44v6 modulates cell proliferation, apoptosis, angiogenesis and therapeutic responses via the PI3K/Akt/mTOR signalling pathway To investigate whether the PI3K/Akt/mTOR signalling pathway is involved in the effects of CD44v6 KD on CaP, several signalling pathway proteins were examined on sections from s.c. mouse xenografts by IHC.
10
The results from IHC again confirmed the KD of CD44v6 in the xenografts (Fig.4a, Table S3), and along with this, it was demonstrated that the phosphorylated PI3K/Akt/mTOR signalling proteins (p-mTOR and p-Akt) were downregulated compared to PC-3M-CD44v6scr group, whereas no noticeable changes were seen in total mTOR and Akt (Fig.4b-e, Table S3). To further investigate whether KD of CD44v6 affects proliferative potential in primary CaP tumours, tumour sections from s.c. xenografts were assessed for Ki-67 expression. Strong expression of Ki-67 was found in PC-3M-CD44v6-scr xenografts while a weak expression of Ki-67 was observed in PC-3M-CD44v6-KD xenografts (Fig.4f, Table S3), suggesting that CD44v6 is associated with the proliferation of CaP in vivo. Active Caspase-3 and CD31 were examined to assess the tumour response to DTX, and γH2AX was examined to assess the tumour response to RT. Increased Caspase-3 (active) and decreased CD31 expression were found in PC-3M-CD44v6-KD CaP xenografts compared to PC-3MCD44v6-scr group (Fig. 4g,h, Table S3), suggesting that upregulated apoptosis (active Caspase-3) and reduced angiogenic activity (CD31) were found in response to DTX treatment after KD of CD44v6. Later, we found the DNA double-strand breaks marker γH2AX expression was increased in PC-3M-CD44v6-KD xenografts compared to PC-3MCD44v6-scr ones (Fig. 4i, Table S3), suggesting that sensitisation of CaP to RT could be modulated by CD44v6 in vivo. These data indicate that KD of CD44v6 is associated with cell proliferation, apoptosis, angiogenesis and therapeutic responses in vivo, possibly via regulation of the PI3K/Akt/mTOR signalling pathway.
4. Discussion
11
Most of studies on CD44v6 are limited to its expression on cancerous and benign tissues, and whether it is a cancer prognostic marker [11, 12]. In CaP, whether the expression of CD44v6 has a relationship with prognosis is still inconclusive, with studies showing a positive relation [13], no relation [14] or an adverse relation [15]. There are even few data available to demonstrate its role in CaP progression and metastasis, chemo-/radioresistance, least of all the underlying signalling pathways involved. In the previous study [8], we demonstrated that the overexpression of CD44v6 was found in seven metastatic CaP cell lines and on human primary CaP tissues and lymph node metastases, rather than normal prostate cells and tissues. KD of CD44v6 suppressed CaP proliferative, invasive and adhesive capacity, reduced sphere formation, enhanced chemo/radiosensitivity and downregulated epithelial-mesenchymal transition (EMT), PI3K/Akt, Wnt/β-catenin pathways. To better mimic the clinical settings and more reliably test its effect on chemo-/radiosensitivity, in this study we move forward by using a CD44v6-KD NOD/SCID mouse model. We established s.c. and orthotopic CaP xenografts to investigate the roles of CD44v6 in tumour growth, chemo-/radiosensitivity and the association of the PI3K/Akt/mTOR signalling pathway in vivo. It was found that CD44v6 is actively involved in CaP tumourigenesis and chemo-/radioresistance. To the best of our knowledge, this is the first study to investigate the roles of CD44v6 in CaP development and therapeutic resistance and associated signalling pathway in vivo. In this study, it showed that KD of CD44v6 can significantly reduce tumour growth in both s.c. and orthotopic mouse models, respectively. The results are in line with our previous in vitro study [8]. CD44 molecule functions as a cellular adhesion molecule for hyaluronic acid (HA) which is a major component of extracellular matrix (ECM). ECM provides important cues to direct cell growth and motility. Moreover, the interaction of the anti-adhesion molecules with HA and CD44 promotes the expansion of the pericellular matrix. These
12
complexes increase the viscoelastic nature of the pericellular matrix, creating a highly malleable extracellular environment that supports cell shape changes, which are necessary for cancer cell proliferation and migration [16]. Lourenco et al. found that in the context of gastric cancer, CD44v6 could modulate ECM remodelling and enhance the malignant potential of gastric cancer cells [17]. KD of CD44v6 can potentially decrease the binding affinity or capacity of tumour cells to ECM, which results in a reduction of tumour growth and progression. It was also found that CD44v6 drove tumour invasion in oral cancer, which was primarily associated with the activation of PI3K/Akt pathway [18], which regulates cell proliferation, malignant transformation and metastasis [19]. In the current study, KD of CD44v6 significantly sensitised CaP xenografts to DTX. P53 was found to be able to counteract CD44-mediated anti-apoptosis via binding to a noncanonical sequence in the CD44 promoter [20], however, the p53 gene is mutated [TP53 p.Lys139fs*31 (c.413delC)] in PC-3M cell line which we used to establish the mouse models, bestowing the cells an intrinsic resistance to apoptosis. As previously reviewed [21], the multidrug resistance (MDR) gene participates in the chemoresistance of CaP, and it was reported that the MDR could be enhanced by HA-CD44v6 interaction, via upregulating PI3K/Akt pathway [22]. Moreover, several other groups also reported that inhibiting the PI3K/Akt/mTOR signalling pathway enhanced epirubicin, cisplatin, DTX and other chemotherapy treatments [23]. Consistent with these findings, our results showed that KD of CD44v6 is associated with downregulated PI3K/Akt/mTOR pathway and improved chemosensitivity in CaP. Overexpression of CD44v6 contributes to radioresistance of CaP. Chang et al. found overexpression of CD44v6 was correlated with radioresistance of three CaP-radioresistant metastatic cell lines [24], and KD of CD44v6 enhanced the radiosensitivity of CaP cells by reversing EMT in vitro [8]. Choi et al. also found that targeting radiation-induced
13
endothelial-mesenchymal transition attenuated the reactivation of CD44v6+ cancer cells and enhanced RT efficacy [25]. It was believed that the PI3K/Akt/mTOR pathway was also one of the major players in the acquisition of radioresistant phenotype. Liu et al. reported that inhibition of the PI3K/Akt/mTOR pathway suppressed tumour progression and increased radiosensitivity in nasopharyngeal carcinoma [26]. Chang et al. found that inhibiting PI3K/Akt/mTOR pathway enhanced radiosensitivity in radioresistant CaP cells through inducing apoptosis, reducing autophagy, suppressing DNA repair pathways [27]. Our results showed that KD of CD44v6 could significantly radiosensitise the tumour, supported by increased DNA double-strand break marker γH2AX, implying the important role of CD44v6 in regulating CaP radiosensitivity. Additionally, it was recently reported that hypoxia within the tumour led to an upregulated expression of CD44v6 accompanied by an increase in the radioresistance in colorectal cancers [28]. Krishnamachary et al. also demonstrated hypoxic tumour regions were associated with high expression of CD44v6, but did not evaluate the effects on radiosensitivity [29]. Further investigations are warranted to elucidate the regulatory role of CD44v6 in radiosensitivity with other pathways, such as NF-κB and IL6/STAT3 pathways [30, 31]. Over the past decade, CD44 (in its standard isoform) has been well documented as a common cancer stem cell (CSC) marker in many cancers including CaP [32], however, very little attention has been paid to CD44v6 in CaP. In colorectal cancer, increased CD44v6 expression were found in all colorectal CSCs, and inhibition of PI3K selectively eradicated CD44v6+ CSCs and reduced metastasis [33]. Targeting CD44v6 with 5FU and Silibinin could attenuate the stemness of colorectal CSCs, possibly via dual inhibition of PI3K/MAPK pathways [34]. More interestingly, CD44v6 was recently found to prominently contribute to the exosome-mediated reprogramming of noncancer-initiating cells to cancer-initiating cells in pancreatic cancer [35].
14
As it plays an important role in CaP tumourigenesis and therapeutic resistance, and its expression is exclusively found in cancerous tissues, CD44v6 has a potential to serve as an ideal candidate for targeted therapy in CaP treatment. Current CD44v6-targeted therapies include anti-CD44v6 antibody, anti-CD44v6
antibody-conjugated
chemo drug or
radioisotope, and HA/CD44v6 interaction perturbation [36]. Casucci et al. found that CD44v6-targeted T cells possessed potent antitumour effects against primary human acute myeloid leukaemia and multiple myeloma cells while sparing normal hematopoietic stem cells and CD44v6-expressing keratinocytes [37]. Leuci et al. proposed CD44v6 as an innovative target in chimeric antigen receptor (CAR) redirected cytokine-induced killer cells in the treatment of high-grade soft tissue sarcoma [38], opening a new field for the usage of CD44v6 in cancer immunotherapy. In addition, combination of CD44v6-targeting agents and PI3K/Akt/mTOR inhibitors with radiothearapy or chemotherapy is worthy of consideration for recurrent CaP in the future. Taken together, we have demonstrated that CD44v6 is closely involved in CaP tumour growth, chemo-/radiosensitivity and associated with the activation of PI3K/Akt/mTOR pathway in vivo. Our findings shed new light on a potential role of CD44v6 in CaP progression, therapeutic resistance and maintenance of prostate CSCs, and lead to an expansion of the repertoire of therapeutic strategies for CaP patients.
Author Contributions JN, BC, WD and YL conceived and designed the experiments. JN carried out all cellular and animal experiments and analyzed the data. BC provided assistance in shRNA knockdown. JB (Julia Beretov) provided assistance in animal tissue processing, immunohistochemistry and assessment in immunostaning. JN and YL wrote the manuscript. JB (Joseph Bucci), DM and PG reviewed the data and manuscript. All authors edited and approved the manuscript.
15
Conflict of Interest The authors declare no conflict of interest.
Acknowledgments This study was supported in part by Cancer Research Trust Fund at Cancer Care Centre, St. George Hospital, China Scholarship Council (CSC) and Prostate and Breast Cancer Foundation (PBCF). The funding sources listed above had no role in study design; collection, management, analysis and interpretation of data; the decision to publish or preparation of the manuscript. The authors received no specific funding for this work. We also thank our research team members for their support in the animal experiments.
References [1]
R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2018, CA Cancer J Clin 68
(2018) 7-30. [2]
D.A. Palacios, M. Miyake, C.J. Rosser, Radiosensitization in prostate cancer:
mechanisms and targets, BMC urology 13 (2013) 4. [3]
I.F. Tannock, R. de Wit, W.R. Berry, J. Horti, A. Pluzanska, K.N. Chi, S. Oudard, C.
Theodore, N.D. James, I. Turesson, M.A. Rosenthal, M.A. Eisenberger, T.A.X. Investigators, Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer, The New England journal of medicine 351 (2004) 1502-1512. [4]
D.P. Petrylak, C.M. Tangen, M.H. Hussain, P.N. Lara, Jr., J.A. Jones, M.E. Taplin,
P.A. Burch, D. Berry, C. Moinpour, M. Kohli, M.C. Benson, E.J. Small, D. Raghavan, E.D. Crawford, Docetaxel and estramustine compared with mitoxantrone and prednisone for 16
advanced refractory prostate cancer, The New England journal of medicine 351 (2004) 15131520. [5]
A.M. Afify, S. Tate, B. Durbin-Johnson, D.M. Rocke, T. Konia, Expression of CD44s
and CD44v6 in lung cancer and their correlation with prognostic factors, The International journal of biological markers 26 (2011) 50-57. [6]
J. Odenthal, M. Rijpkema, D. Bos, E. Wagena, H. Croes, R. Grenman, O. Boerman, R.
Takes, P. Friedl, Targeting CD44v6 for fluorescence-guided surgery in head and neck squamous cell carcinoma, Scientific reports 8 (2018) 10467. [7]
A. Afify, P. Purnell, L. Nguyen, Role of CD44s and CD44v6 on human breast cancer
cell adhesion, migration, and invasion, Experimental and molecular pathology 86 (2009) 95100. [8]
J. Ni, P.J. Cozzi, J.L. Hao, J. Beretov, L. Chang, W. Duan, S. Shigdar, W.J. Delprado,
P.H. Graham, J. Bucci, J.H. Kearsley, Y. Li, CD44 variant 6 is associated with prostate cancer metastasis and chemo-/radioresistance, The Prostate 74 (2014) 602-617. [9]
J. Ni, P. Cozzi, J. Beretov, W. Duan, J. Bucci, P. Graham, Y. Li, Epithelial cell
adhesion molecule (EpCAM) is involved in prostate cancer chemotherapy/radiotherapy response in vivo, BMC cancer 18 (2018) 1092. [10]
J. Ni, P. Cozzi, T.T. Hung, J. Hao, P. Graham, Y. Li, Monitoring Prostate Tumor
Growth in an Orthotopic Mouse Model Using Three-Dimensional Ultrasound Imaging Technique, Translational oncology 9 (2016) 41-45. [11]
K. Okuyama, H. Fukushima, T. Naruse, S. Yanamoto, H. Tsuchihashi, M. Umeda,
CD44 Variant 6 Expression and Tumor Budding in the Medullary Invasion Front of Mandibular Gingival Squamous Cell Carcinoma Are Predictive Factors for Cervical Lymph Node Metastasis, Pathology oncology research : POR 25 (2019) 603-609.
17
[12]
H. Kansu-Celik, M. Gungor, F. Ortac, D. Kankaya, A. Ensari, Expression of CD44
variant 6 and its prognostic value in benign and malignant endometrial tissue, Archives of gynecology and obstetrics 296 (2017) 313-318. [13]
H. Gu, P. Shang, C. Zhou, [Expression of CD44v6 and E-cadherin in prostate
carcinoma and metastasis of prostate carcinoma], Zhonghua nan ke xue = National journal of andrology 10 (2004) 32-34, 38. [14]
S. Ekici, W.H. Cerwinka, R. Duncan, P. Gomez, F. Civantos, M.S. Soloway, V.B.
Lokeshwar, Comparison of the prognostic potential of hyaluronic acid, hyaluronidase (HYAL-1), CD44v6 and microvessel density for prostate cancer, International journal of cancer. Journal international du cancer 112 (2004) 121-129. [15]
S. Aaltomaa, P. Lipponen, M. Ala-Opas, V.M. Kosma, Expression and prognostic
value of CD44 standard and variant v3 and v6 isoforms in prostate cancer, European urology 39 (2001) 138-144. [16]
S.P. Evanko, M.I. Tammi, R.H. Tammi, T.N. Wight, Hyaluronan-dependent
pericellular matrix, Advanced drug delivery reviews 59 (2007) 1351-1365. [17]
B.N. Lourenco, N.L. Springer, D. Ferreira, C. Oliveira, P.L. Granja, C. Fischbach,
CD44v6 increases gastric cancer malignant phenotype by modulating adipose stromal cellmediated ECM remodeling, Integrative biology : quantitative biosciences from nano to macro 10 (2018) 145-158. [18]
T. Kashyap, K.K. Pramanik, N. Nath, P. Mishra, A.K. Singh, S. Nagini, A. Rana, R.
Mishra, Crosstalk between Raf-MEK-ERK and PI3K-Akt-GSK3beta signaling networks promotes chemoresistance, invasion/migration and stemness via expression of CD44 variants (v4 and v6) in oral cancer, Oral oncology 86 (2018) 234-243.
18
[19]
L. Chang, P.H. Graham, J. Ni, J. Hao, J. Bucci, P.J. Cozzi, Y. Li, Targeting
PI3K/Akt/mTOR signaling pathway in the treatment of prostate cancer radioresistance, Critical reviews in oncology/hematology 96 (2015) 507-517. [20]
C. Chen, S. Zhao, A. Karnad, J.W. Freeman, The biology and role of CD44 in cancer
progression: therapeutic implications, J Hematol Oncol 11 (2018) 64. [21]
J. Ni, P. Cozzi, J. Hao, W. Duan, P. Graham, J. Kearsley, Y. Li, Cancer stem cells in
prostate cancer chemoresistance, Current cancer drug targets 14 (2014) 225-240. [22]
S. Misra, S. Ghatak, B.P. Toole, Regulation of MDR1 expression and drug resistance
by a positive feedback loop involving hyaluronan, phosphoinositide 3-kinase, and ErbB2, The Journal of biological chemistry 280 (2005) 20310-20315. [23]
F. Ji, D. Ma, Z. Liu, X. Xie, Rosiglitazone amplifies the sensitivity of docetaxel and
reduces the expression of CD44v6, Oncology letters 7 (2014) 1284-1288. [24]
L. Chang, P.H. Graham, J. Hao, J. Ni, J. Bucci, P.J. Cozzi, J.H. Kearsley, Y. Li,
Acquisition of epithelial-mesenchymal transition and cancer stem cell phenotypes is associated with activation of the PI3K/Akt/mTOR pathway in prostate cancer radioresistance, Cell death & disease 4 (2013) e875. [25]
S.H. Choi, A.R. Kim, J.K. Nam, J.M. Kim, J.Y. Kim, H.R. Seo, H.J. Lee, J. Cho, Y.J.
Lee, Tumour-vasculature development via endothelial-to-mesenchymal transition after radiotherapy controls CD44v6(+) cancer cell and macrophage polarization, Nature communications 9 (2018) 5108. [26]
T. Liu, Q. Sun, Q. Li, H. Yang, Y. Zhang, R. Wang, X. Lin, D. Xiao, Y. Yuan, L.
Chen, W. Wang, Dual PI3K/mTOR inhibitors, GSK2126458 and PKI-587, suppress tumor progression and increase radiosensitivity in nasopharyngeal carcinoma, Molecular cancer therapeutics 14 (2015) 429-439.
19
[27]
L. Chang, P.H. Graham, J. Hao, J. Ni, J. Bucci, P.J. Cozzi, J.H. Kearsley, Y. Li,
PI3K/Akt/mTOR pathway inhibitors enhance radiosensitivity in radioresistant prostate cancer cells through inducing apoptosis, reducing autophagy, suppressing NHEJ and HR repair pathways, Cell death & disease 5 (2014) e1437. [28]
G. Wohlleben, K. Hauff, M. Gasser, A.M. Waaga-Gasser, T. Grimmig, M. Flentje, B.
Polat, Hypoxia induces differential expression patterns of osteopontin and CD44 in colorectal carcinoma, Oncol Rep 39 (2018) 442-448. [29]
B. Krishnamachary, M.F. Penet, S. Nimmagadda, Y. Mironchik, V. Raman, M.
Solaiyappan, G.L. Semenza, M.G. Pomper, Z.M. Bhujwalla, Hypoxia regulates CD44 and its variant isoforms through HIF-1alpha in triple negative breast cancer, PloS one 7 (2012) e44078. [30]
Y.Y. Xu, M. Guo, L.Q. Yang, F. Zhou, C. Yu, A. Wang, T.H. Pang, H.Y. Wu, X.P.
Zou, W.J. Zhang, L. Wang, G.F. Xu, Q. Huang, Regulation of CD44v6 expression in gastric carcinoma by the IL-6/STAT3 signaling pathway and its clinical significance, Oncotarget 8 (2017) 45848-45861. [31]
S. Damm, P. Koefinger, M. Stefan, C. Wels, G. Mehes, E. Richtig, H. Kerl, M. Otte,
H. Schaider, HGF-promoted motility in primary human melanocytes depends on CD44v6 regulated via NF-kappa B, Egr-1, and C/EBP-beta, The Journal of investigative dermatology 130 (2010) 1893-1903. [32]
Hao JL, Ni J, Graham P, Cozzi P, Bucci J, Kearsley J, L. Yi, The CD44 isoforms in
prostate cancer metastasis and progression, World Journal of Cancer Research 1 (2013) 3-14. [33]
M. Todaro, M. Gaggianesi, V. Catalano, A. Benfante, F. Iovino, M. Biffoni, T.
Apuzzo, I. Sperduti, S. Volpe, G. Cocorullo, G. Gulotta, F. Dieli, R. De Maria, G. Stassi, CD44v6 is a marker of constitutive and reprogrammed cancer stem cells driving colon cancer metastasis, Cell stem cell 14 (2014) 342-356.
20
[34]
S. Patel, B. Waghela, K. Shah, F. Vaidya, S. Mirza, S. Patel, C. Pathak, R. Rawal,
Silibinin, A Natural Blend In Polytherapy Formulation For Targeting Cd44v6 Expressing Colon Cancer Stem Cells, Scientific reports 8 (2018) 16985. [35]
Z. Wang, H. Sun, J. Provaznik, T. Hackert, M. Zoller, Pancreatic cancer-initiating cell
exosome message transfer into noncancer-initiating cells: the importance of CD44v6 in reprogramming, Journal of experimental & clinical cancer research : CR 38 (2019) 132. [36]
S. Misra, P. Heldin, V.C. Hascall, N.K. Karamanos, S.S. Skandalis, R.R. Markwald, S.
Ghatak, Hyaluronan-CD44 interactions as potential targets for cancer therapy, The FEBS journal 278 (2011) 1429-1443. [37]
M. Casucci, B. Nicolis di Robilant, L. Falcone, B. Camisa, M. Norelli, P. Genovese,
B. Gentner, F. Gullotta, M. Ponzoni, M. Bernardi, M. Marcatti, A. Saudemont, C. Bordignon, B. Savoldo, F. Ciceri, L. Naldini, G. Dotti, C. Bonini, A. Bondanza, CD44v6-targeted T cells mediate potent antitumor effects against acute myeloid leukemia and multiple myeloma, Blood 122 (2013) 3461-3472. [38]
V. Leuci, G.M. Casucci, G. Grignani, R. Rotolo, U. Rossotti, E. Vigna, L.
Gammaitoni, G. Mesiano, E. Fiorino, C. Donini, A. Pisacane, L.D. Ambrosio, Y. Pignochino, M. Aglietta, A. Bondanza, D. Sangiolo, CD44v6 as innovative sarcoma target for CARredirected CIK cells, Oncoimmunology 7 (2018) e1423167.
Figure Legends Fig. 1. Expression of CD44v6 in PC-3M-CD44v6-KD, PC-3M-CD44v6-scr and PC-3M cell lines. (A) WB is shown to confirm the KD of CD44v6 in PC-3M cell line. ß-tubulin was chosen as a loading control. (B) KD of CD44v6 is further confirmed by qRT-PCR. Results are expressed as Mean ± SD (n=3). ***P<0.001. KD, knock-down; scr, scrambled shRNA control; WB, Western blot
21
Fig. 2. Tumour growth of PC-3M-CD44v6-KD and PC-3M-CD44v6-scr xenografts in CaP s.c. and orthotopic mouse models. (A) Graph is shown for tumour growth curves of PC-3MCD44v6-KD and PC-3M-CD44v6-scr cells in the s.c. xenograft model, the weekly measurements of PC-3M-CD44v6-KD xenografts showed a significant reduced tumour growth compared to the PC-3M-CD44v6-scr group (n=9, mean±SD, P<0.05). (B) At the end of the experiment, tumour weight from PC-3M-CD44v6-KD group mice was significantly decreased compared to that from PC-3M-CD44v6-scr group mice in s.c. model (n=9, mean±SD, * indicates P<0.05). (C) Representative images of tumours harvested from s.c. xenografts are shown. (D) Graph is shown for growth curves of PC-3M-CD44v6-KD and PC3M-CD44v6-scr xenografts in the orthotopic xenograft model (n=9, mean±SD, P<0.05). (E) At the end of the experiment, tumour weight from PC-3M-CD44v6-KD group mice was significantly decreased compared to that from PC-3M-CD44v6-scr group mice in orthotopic model (n=9, mean±SD, *P<0.05). (F) Representative images of tumours from orthotopic xenografts are shown. KD, knock-down; scr, scrambled shRNA control.
Fig. 3. In vivo effects of chemo-/radiosensitivity of PC-3M-CD44v6-KD and PC-3MCD44v6-scr cell lines in s.c. animal models. (A) Tumour growth curves are shown for DTX treatment groups in PC-3M-CD44v6-KD and PC-3M-CD44v6-scr s.c. mouse models. (B) The ratios of DTX-treated versus VC-treated tumour volumes (DTX/VC) were calculated and plotted from the start of DTX treatment to the end of experiments in s.c. mouse model. (C) Tumour growth curves are shown for RT groups in PC-3M-CD44v6-KD and PC-3MCD44v6-scr s.c. mouse model. (D) The ratios of RT versus SC tumour volumes (RT/SC) were calculated and plotted from the start of RT to the end of experiments in s.c. mouse model. DTX, docetaxel; KD, knock-down; SC, sham control; RT, radiotherapy; scr, scrambled shRNA control; VC, vehicle control.
Fig. 4. EpCAM, PI3K/mTOR/Akt pathway proteins, Ki-67, CD31, Caspase-3 (active) and γH2AX expressions in s.c. CaP mouse xenografts. (A) Significantly reduced expression of CD44v6 was observed after KD of CD44v6 in the s.c. xenograft. (B-E) Phosphorylated PI3K/Akt/mTOR signalling proteins (p-mTOR and p-Akt) were downregulated in CD44v6KD group compared to CD44v6-scr group, whereas no obvious changes were seen in total mTOR and Akt between the two groups . (F) Strong expression of Ki-67 was observed in PC3M-CD44v6-scr s.c. xenografts while after KD of CD44v6, the expression of Ki-67 is weak. (G) Markedly reduced CD31 expression was seen in PC-3M-CD44v6-KD xenografts 22
compared to PC-3M-CD44v6-scr group after DTX treatment. (H) An increased level of active Caspase-3 expression was seen in PC-3M-CD44v6-KD xenografts compared to PC3M-CD44v6-scr xenografts after treatment with DTX. (I) After RT, an increased level of γH2AX expression was seen in PC-3M-CD44v6-KD xenografts compared to PC-3MCD44v6-scr xenografts. Magnification x400 in all images. DTX, docetaxel; KD, knock-down; RT, radiotherapy; s.c., subcutaneous; scr, scrambled shRNA control.
Fig. S1. Validation of CD44v6-KD using three independent shRNA in PC-3M cell line. WB is shown to confirm the KD of CD44v6 in PC-3M cell line using three independent shRNAs and one scrambled shRNA control. ß-tubulin was chosen as a loading control. No significant difference was observed. KD, knock-down; scr, scrambled shRNA control; shRNA, short hairpin RNA; WB, Western blot.
23
-3 M
-C
PC-3M
PC
PC-3M-CD44v6-KD PC-3M-CD44v6-scr
-C
β-tubulin
-3 M
CD44v6
D 44 v6 -
K
D
sc r
-3 M D 44 v6 -
PC
Normalised CD44v6 Expression (%)
B
PC
A
A
B
C
D
E
F
A
B
C
D
PC-3MPC-3MCD44V6-KD CD44v6-scr
PC-3MCD44V6-KD
PC-3MCD44v6-scr
A
CD44v6
F
Ki-67
B
Akt
G
CD31
C
p-Akt
H
Caspase-3 (active)
D
mTOR
I
γH2AX
E
p-mTOR
COI statement
We have no conflict of interest for this submission.
CD44 variant 6 is associated with prostate cancer growth and chemo/radiotherapy response in vivo Jie Nia,b, Belamy B. Cheungc,d, Julia Beretova,b,e, Wei Duanf, Joseph Buccia,b, David Maloufa,g, Peter Grahama,b, Yong Lia,b,h,*
a
Cancer Care Centre, St George Hospital, Kogarah, NSW 2217, Australia
b
St George and Sutherland Clinical School, Faculty of Medicine, UNSW Sydney, NSW 2052,
Australia c
Children's Cancer Institute Australia, Lowy Cancer Research Centre, UNSW Sydney, NSW
2052, Australia d
School of Women's & Children's Health, Faculty of Medicine, UNSW Sydney, NSW 2052,
Australia e
Anatomical Pathology, NSW Health Pathology, St George Hospital, Kogarah, NSW 2217,
Australia f
School of Medicine and Centre for Molecular and Medical Research, Deakin University, Waurn
Ponds, VIC 3216, Australia g
Department of Urology, St George Hospital, Kogarah NSW 2217, Australia
h
School of Basic Medical Sciences, Zhengzhou University, Henan 450001, China
*Correspondence Yong Li, Cancer Care Centre, St George Hospital, Level 2, 4-10 South St, Kogarah NSW 2217 Australia. Tel: +61-2-91131338; Fax: +61-2-91131386; Email: [email protected]
S0014-4827(20)30044-6
DOI:
https://doi.org/10.1016/j.yexcr.2020.111850
Reference:
YEXCR 111850
To appear in:
Experimental Cell Research
Received Date: 4 October 2019 Revised Date:
8 January 2020
Accepted Date: 13 January 2020
Please cite this article as: J. Ni, B.B. Cheung, J. Beretov, W. Duan, J. Bucci, D. Malouf, P. Graham, Y. Li, CD44 variant 6 is associated with prostate cancer growth and chemo-/radiotherapy response in vivo, Experimental Cell Research (2020), doi: https://doi.org/10.1016/j.yexcr.2020.111850. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
CRediT author statement Jie Ni: Conceptualization, Methodology, Investigation, Formal analysis, Writing - Original Draft, Visualization. Belamy Cheung: Conceptualization, Methodology, Resources. Julia Beretov: Investigation, Formal analysis. Wei Duan: Conceptualization, Methodology. Joseph Bucci: Resources, Writing - Review & Editing. David Malouf: Resources, Writing - Review & Editing. Peter Graham: Resources, Supervision, Writing - Review & Editing, Funding acquisition. Yong Li: Conceptualization, Supervision, Writing - Review & Editing, Funding acquisition.
CD44 variant 6 is associated with prostate cancer growth and chemo/radiotherapy response in vivo Jie Nia,b, Belamy B. Cheungc,d, Julia Beretova,b,e, Wei Duanf, Joseph Buccia,b, David Maloufa,g, Peter Grahama,b, Yong Lia,b,h,*
a
Cancer Care Centre, St George Hospital, Kogarah, NSW 2217, Australia
b
St George and Sutherland Clinical School, Faculty of Medicine, UNSW Sydney, NSW 2052,
Australia c
Children's Cancer Institute Australia, Lowy Cancer Research Centre, UNSW Sydney, NSW
2052, Australia d
School of Women's & Children's Health, Faculty of Medicine, UNSW Sydney, NSW 2052,
Australia e
Anatomical Pathology, NSW Health Pathology, St George Hospital, Kogarah, NSW 2217,
Australia f
School of Medicine and Centre for Molecular and Medical Research, Deakin University,
Waurn Ponds, VIC 3216, Australia g
Department of Urology, St George Hospital, Kogarah NSW 2217, Australia
h
School of Basic Medical Sciences, Zhengzhou University, Henan 450001, China
*Correspondence Yong Li, Cancer Care Centre, St George Hospital, Level 2, 4-10 South St, Kogarah NSW 2217 Australia. Tel: +61-2-91131338; Fax: +61-2-91131386; Email: [email protected]
1
ABSTRACT We have previously demonstrated that CD44 variant 6 (CD44v6) is associated with prostate cancer (CaP) growth and therapeutic resistance in vitro, however, the role of CD44v6 in CaP in vivo is not fully understood. The purpose of this study is to investigate the effect of CD44v6 on CaP growth and chemo−/radiotherapy response in NOD/SCID mouse models in vivo and to validate its role as a therapeutic target for CaP therapy. CD44v6 was knocked down in PC-3M CaP cell line using short hairpin RNA. Subcutaneous (s.c.) and orthotopic CaP mouse xenografts were established. The effect of CD44v6 knockdown (KD) on tumour growth was evaluated on both s.c. and orthotopic models. Chemo−/radiotherapy response was evaluated on the s.c. model. Association of CD44v6 with PI3K/Akt pathway was validated using immunohistochemistry staining. We found that KD of CD44v6 significantly reduced tumour growth in both models, and enhanced the sensitivity of tumours to chemotherapy and radiotherapy in the s.c. model. In addition, we demonstrated that KD of CD44v6 is associated with downregulation of the PI3K/Akt/mTOR pathway. Our data confirm that CaP growth and chemo−/radiosensitivity in vivo is associated with CD44v6, which holds great promises as a therapeutic target in the treatment of CaP.
Keywords CD44v6, prostate cancer, PI3K/Akt/mTOR, chemotherapy, radiotherapy
2
1. Introduction Prostate cancer (CaP) is the most prevalent cancer diagnosed in men in the western world, constituting nearly 20% of new cancer cases in the United States in 2018 [1]. One of the biggest therapeutic challenges of CaP is the development of resistance to radiation and chemotherapy. Radiation therapy, either brachytherapy or external beam radiation therapy, remains a mainstay in the treatment of CaP, particularly for the organ-confined disease. However, a small subset of cancer cells will acquire resistance during fractionated irradiation, and thereby survived the radiation, which leads to locoregional recurrence and distant metastasis [2]. Radioresistance is one of the major factors that limit the efficacy of radiotherapy, and radiosensitisation of cancer cells is of great significance for the CaP radiotherapy. Chemotherapy is the main treatment option for metastatic CaP. Despite extensive research on novel chemotherapy agents for over a decade, docetaxel (DTX) has been used as the firstline agents since 2004, following two Phase III trials [3, 4] where docetaxel demonstrated overall survival benefits in metastatic castration-resistant prostate cancer (mCRPC) patients. However, approximately half of mCRPC patients do not respond, and those who do will eventually develop resistance to docetaxel, posing a significant challenge in the treatment of late-stage CaP. CD44 variant 6 (CD44v6) is one of the isoforms of CD44 by alternative mRNA splicing and has been found to be closely associated with tumour behaviour in various cancers [5-7]. In particular, our previous study demonstrated that CD44v6 is associated with CaP proliferation, invasion and chemo-/radio-resistance in vitro [8]. However, few studies on the roles of CD44v6 in CaP chemo−/radioresistance in vivo are available. To address this, in the current study, we used subcutaneous (s.c.) and orthotopic CaP NOD/SCID mouse models to investigate the effect of CD44v6 on CaP growth and used the s.c. model to test the
3
chemo−/radiotherapy response. We demonstrate for the first time that CD44v6 is involved in tumour growth and chemo−/radiotherapy response in CaP animal models in vivo, and associated with the PI3K/Akt/mTOR pathway activation.
2. Methods 2.1. Antibodies and reagents Detailed information and conditions of antibodies used in the study are listed in Table S1. DTX was purchased from Hospira (Melbourne, VIC, Australia), and diluted in 0.9% saline for in vitro study. All cell culture reagents were purchased from Life Technologies Australia Pty Ltd (Melbourne, VIC, Australia) unless otherwise stated. MISSION® lentiviral transduction particles encoding for short hairpin RNA (shRNA) against CD44v6 and a nontarget shRNA control (scr) transduction particles were designed and obtained from SigmaAldrich Pty Ltd (Castle Hill, NSW, Australia). 2.2. Cell line and cell culture Androgen-non-responsive PC-3M CaP cell line was obtained from MD Anderson Cancer Center under Material Transfer Agreement and cultured in RPMI-1640 supplemented with 10% (vol/vol) FBS, 50 U/mL of penicillin, and 50 µg/mL of streptomycin. The cells were maintained in a humidified incubator at 37 °C and 5% CO2. The identity of PC-3M was confirmed by short tandem repeat profiling (CellBank Australia, NSW, Australia) and the assays were performed within a few passages of initial authentication. The cell line was regularly tested to confirm the absence of mycoplasma contamination using the LookOut® mycoplasma qPCR detection kit (Sigma-Aldrich Pty Ltd, NSW, Australia). 2.3. Short hairpin RNA (shRNA) transfection for CD44v6 PC-3M cells were seeded in 6-well plates, and upon reaching 70–80% confluence, the cells were infected with lentiviral particles lentivirus expressing CD44v6 shRNA or a scrambled 4
sequence. After 48 hours, the stable transduced cells were selected in puromycin-containing medium (500 µg/mL), propagated and finally maintained in medium containing 1 mg/mL puromycin for the following experiments. The KD were performed with three different custom-designed shRNAs that target non-overlapping regions of CD44v6 on PC-3M cell line. Detailed information on CD44v6-shRNAs is included in Table S2. 2.4. Western blot Briefly, 20 ug of extracted proteins were separated on Bis-Tris gels (Life Technologies Pty Ltd, Australia) and blotted onto PVDF membranes (Millipore, USA). The membrane was blocked with 5% bovine serum albumin in Tris-buffered saline with 0.1% Tween 20 and incubated overnight at 4 °C with the indicated primary antibodies (Table S1). Next, the membranes were washed and incubated with goat anti-rabbit/mouse IgG secondary antibody for 1 hour at room temperature. Protein expression was analysed using Clarity Max Western ECL Substrate (Bio-rad Laboratories, Australia) in the ImageQuant LAS4000 system (GE Healthcare, USA). Mouse anti-human β-tubulin monoclonal antibody was used as a loading control. 2.5. Quantitative real-time PCR (qRT-PCR) Total RNA was isolated using the High Pure RNA Isolation Kit (Roche Life Science, NSW, Australia) and 2 µg of total RNA was used to synthesise cDNA using the iScript™ cDNA Synthesis Kit (Bio-rad Laboratories, Australia). qRT-PCR was carried out on the CFX96™ Real-Time PCR Detection System (Bio-rad Laboratories, Australia) in a solution containing cDNA, primer and SsoAdvanced Universal SYBR® Green Supermix (Bio-rad Laboratories, Australia). GAPDH was used as a reference control. 2.6. Establishment of animal models All animal procedures in the study followed the protocols approved by the Animal Care and Ethics Committee (ACEC 14/46A) of UNSW Sydney, Australia.
5
Male, 6–8 weeks old NOD/SCID mice (Animal Resources Centre, WA, Australia) were housed in a group of 3 or 4, in specific pathogen-free facilities and all experiments were performed in a laminar flow cabinet. Mice were kept at least 1 week before experimental manipulation. All mice were monitored daily for health status and the weight was recorded at least twice a week. All mice remained healthy and active during the experiments. For the s.c. xenograft model, 2 × 106 PC-3M-CD44v6-KD or PC-3M-CD44v6-scr CaP cells in 100 µL phosphate-buffered saline (PBS) were implanted subcutaneously in the right rear flank region of mice. Tumour progression was recorded weekly by measurements using a calliper, and tumour volumes were calculated as follows: length×width×height×0.52 (in millimetres) for up to 8 weeks. For the orthotopic xenograft model, 1 × 106 PC-3M-CD44v6-KD or PC-3M-CD44v6-scr CaP cells suspended in 50 µL PBS were injected into the prostatic lobe followed by a lower midline laparotomy as previously described [9]. Starting from the second week after cell inoculation, tumour progression was monitored weekly by 3D-ultrasonography [10]. 2.7. In vivo chemosensitivity assay In this study, we used the s.c. mouse model to evaluate the effect of CD44v6 KD on tumour’s response to docetaxel. The dosage of docetaxel was determined by a maximum tolerated dose (MTD) assay set out in our previous study [9]. When the average tumour size reached 70±10 mm3 in each subgroup, 4 mice/per group (n=4) including PC-3M-CD44v6KD and PC-3M-CD44v6-scr groups were treated with 50 mg/kg DTX once by intraperitoneal injection, and 4 mice/per group (n=4) including PC-3M-CD44v6-KD and PC-3M-CD44v6scr groups were treated with saline as the vehicle control (VC). Tumour growth was calculated by measurements using a calliper. 2.8. In vivo radiosensitivity assay
6
We used s.c. mouse model to evaluate the effect of CD44v6 KD on tumour’s response to radiation. The radiation dosage was determined by a previous MTD assay [9]. When the tumour volume reached 70±10 mm3 in s.c. model, mice were anaesthetised with ketamine and xylazine, and then immobilised in a custom-designed chamber with a lead cover. Mice were placed in the X-RAD 320 Biological Irradiator (Precision X-Ray, North Branford, CT, USA) and irradiated with only the tumour area exposed. In each subgroup, 4 mice (n=4) were irradiated at a dose of 2Gy for 4 consecutive days, while 4 mice (n=4) went through the same procedure with the no irradiation as a sham control (SC). 2.9. Immunohistochemistry (IHC) IHC was performed on biological triplicate. Paraffin sections were de-paraffinised and rehydrated, followed by antigen retrieval in DAKO Low pH Target Retrieval Solution (Agilent, VIC, Australia) and then incubated with primary antibodies overnight at 4 °C. For CD31 staining, cryosections were fixed with ice-cold acetone for 10 min, and rinsed in Trisbuffered saline (TBS), before incubated with the CD31 primary antibody. After washing with TBS, slides were then incubated with HRP-conjugated secondary antibody (1:150 dilution) for 45 min at room temperature. Immunoreactivity was then developed with the DAKO Liquid DAB+ Kit (Agilent, VIC, Australia). Slides were then counterstained with Harris Haematoxylin (Thermo Fisher Pty Ltd, VIC, Australia) for 1 minute, and blued with Scott’s Bluing solution (Sigma-Aldrich Pty Ltd, NSW, Australia). Tissue sections were washed in water, dehydrated, cleared and mounted. 2.10. Assessment of immunostaining IHC intensity was assessed using a light microscope (Leica, Germany). The percentage of the tumour cells with positive staining was assessed. Staining intensity was graded between 0-3, following the criteria as follows: - (negative, 0–10%); 1+ (weak, 10–45%); 2+ (moderate,
7
45–70%); 3+ (strong, >70%) of the tumour tissue stained. The evaluation of staining intensity was done independently by two experienced observers (JN and JBeretov). 2.11. Statistical analysis All numerical data were expressed as the average of the values obtained, and the SD was calculated (mean ± SD). Data from different groups were compared using the two-tail student’s t-test. P<0.05 was considered significant. All numerical statistical analyses were performed using the GraphPad Prism 7 (GraphPad, CA, USA).
3. Results 3.1. Expression of CD44v6 in CD44v6-KD and control cells CD44v6 KD was confirmed using Western Blot and qRT-PCR. The KD were performed with three different custom-designed shRNAs that target non-overlapping regions of CD44v6, and after validation, CD44v6-shRNA#1 was selected for the following studies (Fig. S1). As shown in Fig. 1, following shRNA transfection, a reduction of CD44v6 expression on PC3M-CD44v6-KD cells were observed, compared to PC-3M-CD44v6-scr and PC-3M cell lines. No significant difference in the expression of CD44v6 was noted between PC-3MCD44v6-scr and PC-3M cell lines. 3.2. KD of CD44v6 downregulated tumour growth in both s.c. and orthotopic xenografts Weekly measurements of tumour size were plotted. As shown in Fig. 2, both s.c. and orthotopic PC-3M-CD44v6-KD xenografts showed a significant decreased tumour growing pattern compared to the PC-3M-CD44v6-scr group (n=9; P<0.05), respectively. In the s.c. model, all PC-3M-CD44v6-scr xenograft tumours became palpable from the second week post cell inoculation while all tumours from PC-3M-CD44v6-KD group became palpable from the third week after cell inoculation. Significant tumour growth difference was found 3 weeks post cell inoculation (Mean±SD: KD, 15.4±3.9 mm3; scr, 38.0±12.0 mm3; 8
P<0.05). At the end of the experiment (7 weeks after cell inoculation), tumours were harvested, measured and weighed. In PC-3M-CD44v6-KD
group, tumour volume was
260.6±21.5 mm3, tumour weight was 0.6±0.2g; whereas in PC-3M-CD44v6-scr group, tumour volume was 566.1±56.7 mm3, tumour weight was 1.9±0.2g (Mean±SD, P<0.001 in both measurements). In the orthotopic model, the prostatic tumour became visible from the second week post cell inoculation in both PC-3M-CD44v6-KD and PC-3M-CD44v6-scr groups. Significant tumour growth difference was found 3 weeks post cell inoculation (Mean±SD: KD, 42.9±3.3 mm3; scr, 58.9±2.6mm3; P<0.01). After 5 weeks post cell inoculation, in PC-3M-CD44v6KD group, tumour volume was 100.9±12.0 mm3, while in PC-3M-CD44v6-scr group, tumour volume was 231.2±26.0 mm3 (Mean±SD, P<0.01). At the end of the experiment (7 weeks after cell inoculation), tumours were harvested, measured and weighed. In PC-3M-CD44v6KD group, tumour volume was 254.6±37.7 mm3, tumour weight was 0.5±0.2g; whereas in PC-3M-CD44v6-scr group, tumour volume was 231.2±16.0 mm3, tumour weight was 1.7±0.2g (Mean± SD, P<0.01 in both measurements). 3.3. KD of CD44v6 affects CaP chemo-/radio-sensitivity in the s.c. xenograft model DTX or VC treatment was administered at 4 weeks post cell inoculation when the average tumour size reached 70±10 mm3. Tumour volumes were plotted against elapsed time (Fig. 3a). Significant differences were seen between PC-3M-CD44v6-KD-DTX and PC-3MCD44v6-KD-VC groups (n=4, P<0.01), PC-3M-CD44v6-scr-DTX and PC-3M-CD44v6-scrVC groups (n=4, P<0.01), and PC-3M-CD44v6-scr-DTX and PC-3M-CD44v6-scr-VC groups (n=4, P<0.05), respectively. The ratios of DTX-treated versus VC-treated tumour volumes (DTX/VC) were calculated and plotted from the start of DTX treatment to the end of experiments (Fig. 3b). Two groups responded to DTX treatment as early as 1 week posttreatment. Significant differences between two groups in response to DTX were first
9
observed 1 week post DTX treatment. Overall, the ratio of PC-3M-CD44v6-KD tumour volumes decreased faster in response to DTX, compared to the PC-3M-CD44v6-scr group (P<0.01). Notably, in PC-3M-CD44v6-KD, tumours on 75% (3 out of 4) mice became completely impalpable at the end of the experiment, while all PC-3M-CD44v6-scr tumours are still palpable, suggesting that the KD of CD44v6 can greatly increase the DTX sensitivity of CaP tumours in the s.c. mouse model. Fractionated RT (2Gy x 4 fractions) or sham radiation was given 4-week post cell inoculation when the average tumour size reached 70±10 mm3. Tumour volumes were plotted against elapsed time (Fig. 3c). Significant differences were seen between PC-3M-CD44v6KD-RT and PC-3M-CD44v6-KD-SC groups (n=4, P<0.01), PC-3M-CD44v6-scr-RT and PC-3M-CD44v6-scr-SC groups (n=4, P<0.01),
PC-3M-CD44v6-scr-SC and PC-3M-
CD44v6-scr-SC groups (n=4, P<0.05), respectively. The ratios of RT versus SC tumour volumes (RT/SC) were calculated and plotted from the start of RT to the end of experiments (Fig. 3d). PC-3M xenografts responded to RT from 1 week post-treatment. However, more significant differences between the two groups in response to RT were observed 3 weeks post-RT. Overall, the ratio of PC-3M-CD44v6-KD tumour volumes decreased faster in response to RT compared to the sham irradiation (P<0.05), suggesting that the KD of CD44v6 can increase the radiosensitivity of CaP tumours in the s.c. mouse model. 3.4. KD of CD44v6 modulates cell proliferation, apoptosis, angiogenesis and therapeutic responses via the PI3K/Akt/mTOR signalling pathway To investigate whether the PI3K/Akt/mTOR signalling pathway is involved in the effects of CD44v6 KD on CaP, several signalling pathway proteins were examined on sections from s.c. mouse xenografts by IHC.
10
The results from IHC again confirmed the KD of CD44v6 in the xenografts (Fig.4a, Table S3), and along with this, it was demonstrated that the phosphorylated PI3K/Akt/mTOR signalling proteins (p-mTOR and p-Akt) were downregulated compared to PC-3M-CD44v6scr group, whereas no noticeable changes were seen in total mTOR and Akt (Fig.4b-e, Table S3). To further investigate whether KD of CD44v6 affects proliferative potential in primary CaP tumours, tumour sections from s.c. xenografts were assessed for Ki-67 expression. Strong expression of Ki-67 was found in PC-3M-CD44v6-scr xenografts while a weak expression of Ki-67 was observed in PC-3M-CD44v6-KD xenografts (Fig.4f, Table S3), suggesting that CD44v6 is associated with the proliferation of CaP in vivo. Active Caspase-3 and CD31 were examined to assess the tumour response to DTX, and γH2AX was examined to assess the tumour response to RT. Increased Caspase-3 (active) and decreased CD31 expression were found in PC-3M-CD44v6-KD CaP xenografts compared to PC-3MCD44v6-scr group (Fig. 4g,h, Table S3), suggesting that upregulated apoptosis (active Caspase-3) and reduced angiogenic activity (CD31) were found in response to DTX treatment after KD of CD44v6. Later, we found the DNA double-strand breaks marker γH2AX expression was increased in PC-3M-CD44v6-KD xenografts compared to PC-3MCD44v6-scr ones (Fig. 4i, Table S3), suggesting that sensitisation of CaP to RT could be modulated by CD44v6 in vivo. These data indicate that KD of CD44v6 is associated with cell proliferation, apoptosis, angiogenesis and therapeutic responses in vivo, possibly via regulation of the PI3K/Akt/mTOR signalling pathway.
4. Discussion
11
Most of studies on CD44v6 are limited to its expression on cancerous and benign tissues, and whether it is a cancer prognostic marker [11, 12]. In CaP, whether the expression of CD44v6 has a relationship with prognosis is still inconclusive, with studies showing a positive relation [13], no relation [14] or an adverse relation [15]. There are even few data available to demonstrate its role in CaP progression and metastasis, chemo-/radioresistance, least of all the underlying signalling pathways involved. In the previous study [8], we demonstrated that the overexpression of CD44v6 was found in seven metastatic CaP cell lines and on human primary CaP tissues and lymph node metastases, rather than normal prostate cells and tissues. KD of CD44v6 suppressed CaP proliferative, invasive and adhesive capacity, reduced sphere formation, enhanced chemo/radiosensitivity and downregulated epithelial-mesenchymal transition (EMT), PI3K/Akt, Wnt/β-catenin pathways. To better mimic the clinical settings and more reliably test its effect on chemo-/radiosensitivity, in this study we move forward by using a CD44v6-KD NOD/SCID mouse model. We established s.c. and orthotopic CaP xenografts to investigate the roles of CD44v6 in tumour growth, chemo-/radiosensitivity and the association of the PI3K/Akt/mTOR signalling pathway in vivo. It was found that CD44v6 is actively involved in CaP tumourigenesis and chemo-/radioresistance. To the best of our knowledge, this is the first study to investigate the roles of CD44v6 in CaP development and therapeutic resistance and associated signalling pathway in vivo. In this study, it showed that KD of CD44v6 can significantly reduce tumour growth in both s.c. and orthotopic mouse models, respectively. The results are in line with our previous in vitro study [8]. CD44 molecule functions as a cellular adhesion molecule for hyaluronic acid (HA) which is a major component of extracellular matrix (ECM). ECM provides important cues to direct cell growth and motility. Moreover, the interaction of the anti-adhesion molecules with HA and CD44 promotes the expansion of the pericellular matrix. These
12
complexes increase the viscoelastic nature of the pericellular matrix, creating a highly malleable extracellular environment that supports cell shape changes, which are necessary for cancer cell proliferation and migration [16]. Lourenco et al. found that in the context of gastric cancer, CD44v6 could modulate ECM remodelling and enhance the malignant potential of gastric cancer cells [17]. KD of CD44v6 can potentially decrease the binding affinity or capacity of tumour cells to ECM, which results in a reduction of tumour growth and progression. It was also found that CD44v6 drove tumour invasion in oral cancer, which was primarily associated with the activation of PI3K/Akt pathway [18], which regulates cell proliferation, malignant transformation and metastasis [19]. In the current study, KD of CD44v6 significantly sensitised CaP xenografts to DTX. P53 was found to be able to counteract CD44-mediated anti-apoptosis via binding to a noncanonical sequence in the CD44 promoter [20], however, the p53 gene is mutated [TP53 p.Lys139fs*31 (c.413delC)] in PC-3M cell line which we used to establish the mouse models, bestowing the cells an intrinsic resistance to apoptosis. As previously reviewed [21], the multidrug resistance (MDR) gene participates in the chemoresistance of CaP, and it was reported that the MDR could be enhanced by HA-CD44v6 interaction, via upregulating PI3K/Akt pathway [22]. Moreover, several other groups also reported that inhibiting the PI3K/Akt/mTOR signalling pathway enhanced epirubicin, cisplatin, DTX and other chemotherapy treatments [23]. Consistent with these findings, our results showed that KD of CD44v6 is associated with downregulated PI3K/Akt/mTOR pathway and improved chemosensitivity in CaP. Overexpression of CD44v6 contributes to radioresistance of CaP. Chang et al. found overexpression of CD44v6 was correlated with radioresistance of three CaP-radioresistant metastatic cell lines [24], and KD of CD44v6 enhanced the radiosensitivity of CaP cells by reversing EMT in vitro [8]. Choi et al. also found that targeting radiation-induced
13
endothelial-mesenchymal transition attenuated the reactivation of CD44v6+ cancer cells and enhanced RT efficacy [25]. It was believed that the PI3K/Akt/mTOR pathway was also one of the major players in the acquisition of radioresistant phenotype. Liu et al. reported that inhibition of the PI3K/Akt/mTOR pathway suppressed tumour progression and increased radiosensitivity in nasopharyngeal carcinoma [26]. Chang et al. found that inhibiting PI3K/Akt/mTOR pathway enhanced radiosensitivity in radioresistant CaP cells through inducing apoptosis, reducing autophagy, suppressing DNA repair pathways [27]. Our results showed that KD of CD44v6 could significantly radiosensitise the tumour, supported by increased DNA double-strand break marker γH2AX, implying the important role of CD44v6 in regulating CaP radiosensitivity. Additionally, it was recently reported that hypoxia within the tumour led to an upregulated expression of CD44v6 accompanied by an increase in the radioresistance in colorectal cancers [28]. Krishnamachary et al. also demonstrated hypoxic tumour regions were associated with high expression of CD44v6, but did not evaluate the effects on radiosensitivity [29]. Further investigations are warranted to elucidate the regulatory role of CD44v6 in radiosensitivity with other pathways, such as NF-κB and IL6/STAT3 pathways [30, 31]. Over the past decade, CD44 (in its standard isoform) has been well documented as a common cancer stem cell (CSC) marker in many cancers including CaP [32], however, very little attention has been paid to CD44v6 in CaP. In colorectal cancer, increased CD44v6 expression were found in all colorectal CSCs, and inhibition of PI3K selectively eradicated CD44v6+ CSCs and reduced metastasis [33]. Targeting CD44v6 with 5FU and Silibinin could attenuate the stemness of colorectal CSCs, possibly via dual inhibition of PI3K/MAPK pathways [34]. More interestingly, CD44v6 was recently found to prominently contribute to the exosome-mediated reprogramming of noncancer-initiating cells to cancer-initiating cells in pancreatic cancer [35].
14
As it plays an important role in CaP tumourigenesis and therapeutic resistance, and its expression is exclusively found in cancerous tissues, CD44v6 has a potential to serve as an ideal candidate for targeted therapy in CaP treatment. Current CD44v6-targeted therapies include anti-CD44v6 antibody, anti-CD44v6
antibody-conjugated
chemo drug or
radioisotope, and HA/CD44v6 interaction perturbation [36]. Casucci et al. found that CD44v6-targeted T cells possessed potent antitumour effects against primary human acute myeloid leukaemia and multiple myeloma cells while sparing normal hematopoietic stem cells and CD44v6-expressing keratinocytes [37]. Leuci et al. proposed CD44v6 as an innovative target in chimeric antigen receptor (CAR) redirected cytokine-induced killer cells in the treatment of high-grade soft tissue sarcoma [38], opening a new field for the usage of CD44v6 in cancer immunotherapy. In addition, combination of CD44v6-targeting agents and PI3K/Akt/mTOR inhibitors with radiothearapy or chemotherapy is worthy of consideration for recurrent CaP in the future. Taken together, we have demonstrated that CD44v6 is closely involved in CaP tumour growth, chemo-/radiosensitivity and associated with the activation of PI3K/Akt/mTOR pathway in vivo. Our findings shed new light on a potential role of CD44v6 in CaP progression, therapeutic resistance and maintenance of prostate CSCs, and lead to an expansion of the repertoire of therapeutic strategies for CaP patients.
Author Contributions JN, BC, WD and YL conceived and designed the experiments. JN carried out all cellular and animal experiments and analyzed the data. BC provided assistance in shRNA knockdown. JB (Julia Beretov) provided assistance in animal tissue processing, immunohistochemistry and assessment in immunostaning. JN and YL wrote the manuscript. JB (Joseph Bucci), DM and PG reviewed the data and manuscript. All authors edited and approved the manuscript.
15
Conflict of Interest The authors declare no conflict of interest.
Acknowledgments This study was supported in part by Cancer Research Trust Fund at Cancer Care Centre, St. George Hospital, China Scholarship Council (CSC) and Prostate and Breast Cancer Foundation (PBCF). The funding sources listed above had no role in study design; collection, management, analysis and interpretation of data; the decision to publish or preparation of the manuscript. The authors received no specific funding for this work. We also thank our research team members for their support in the animal experiments.
References [1]
R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2018, CA Cancer J Clin 68
(2018) 7-30. [2]
D.A. Palacios, M. Miyake, C.J. Rosser, Radiosensitization in prostate cancer:
mechanisms and targets, BMC urology 13 (2013) 4. [3]
I.F. Tannock, R. de Wit, W.R. Berry, J. Horti, A. Pluzanska, K.N. Chi, S. Oudard, C.
Theodore, N.D. James, I. Turesson, M.A. Rosenthal, M.A. Eisenberger, T.A.X. Investigators, Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer, The New England journal of medicine 351 (2004) 1502-1512. [4]
D.P. Petrylak, C.M. Tangen, M.H. Hussain, P.N. Lara, Jr., J.A. Jones, M.E. Taplin,
P.A. Burch, D. Berry, C. Moinpour, M. Kohli, M.C. Benson, E.J. Small, D. Raghavan, E.D. Crawford, Docetaxel and estramustine compared with mitoxantrone and prednisone for 16
advanced refractory prostate cancer, The New England journal of medicine 351 (2004) 15131520. [5]
A.M. Afify, S. Tate, B. Durbin-Johnson, D.M. Rocke, T. Konia, Expression of CD44s
and CD44v6 in lung cancer and their correlation with prognostic factors, The International journal of biological markers 26 (2011) 50-57. [6]
J. Odenthal, M. Rijpkema, D. Bos, E. Wagena, H. Croes, R. Grenman, O. Boerman, R.
Takes, P. Friedl, Targeting CD44v6 for fluorescence-guided surgery in head and neck squamous cell carcinoma, Scientific reports 8 (2018) 10467. [7]
A. Afify, P. Purnell, L. Nguyen, Role of CD44s and CD44v6 on human breast cancer
cell adhesion, migration, and invasion, Experimental and molecular pathology 86 (2009) 95100. [8]
J. Ni, P.J. Cozzi, J.L. Hao, J. Beretov, L. Chang, W. Duan, S. Shigdar, W.J. Delprado,
P.H. Graham, J. Bucci, J.H. Kearsley, Y. Li, CD44 variant 6 is associated with prostate cancer metastasis and chemo-/radioresistance, The Prostate 74 (2014) 602-617. [9]
J. Ni, P. Cozzi, J. Beretov, W. Duan, J. Bucci, P. Graham, Y. Li, Epithelial cell
adhesion molecule (EpCAM) is involved in prostate cancer chemotherapy/radiotherapy response in vivo, BMC cancer 18 (2018) 1092. [10]
J. Ni, P. Cozzi, T.T. Hung, J. Hao, P. Graham, Y. Li, Monitoring Prostate Tumor
Growth in an Orthotopic Mouse Model Using Three-Dimensional Ultrasound Imaging Technique, Translational oncology 9 (2016) 41-45. [11]
K. Okuyama, H. Fukushima, T. Naruse, S. Yanamoto, H. Tsuchihashi, M. Umeda,
CD44 Variant 6 Expression and Tumor Budding in the Medullary Invasion Front of Mandibular Gingival Squamous Cell Carcinoma Are Predictive Factors for Cervical Lymph Node Metastasis, Pathology oncology research : POR 25 (2019) 603-609.
17
[12]
H. Kansu-Celik, M. Gungor, F. Ortac, D. Kankaya, A. Ensari, Expression of CD44
variant 6 and its prognostic value in benign and malignant endometrial tissue, Archives of gynecology and obstetrics 296 (2017) 313-318. [13]
H. Gu, P. Shang, C. Zhou, [Expression of CD44v6 and E-cadherin in prostate
carcinoma and metastasis of prostate carcinoma], Zhonghua nan ke xue = National journal of andrology 10 (2004) 32-34, 38. [14]
S. Ekici, W.H. Cerwinka, R. Duncan, P. Gomez, F. Civantos, M.S. Soloway, V.B.
Lokeshwar, Comparison of the prognostic potential of hyaluronic acid, hyaluronidase (HYAL-1), CD44v6 and microvessel density for prostate cancer, International journal of cancer. Journal international du cancer 112 (2004) 121-129. [15]
S. Aaltomaa, P. Lipponen, M. Ala-Opas, V.M. Kosma, Expression and prognostic
value of CD44 standard and variant v3 and v6 isoforms in prostate cancer, European urology 39 (2001) 138-144. [16]
S.P. Evanko, M.I. Tammi, R.H. Tammi, T.N. Wight, Hyaluronan-dependent
pericellular matrix, Advanced drug delivery reviews 59 (2007) 1351-1365. [17]
B.N. Lourenco, N.L. Springer, D. Ferreira, C. Oliveira, P.L. Granja, C. Fischbach,
CD44v6 increases gastric cancer malignant phenotype by modulating adipose stromal cellmediated ECM remodeling, Integrative biology : quantitative biosciences from nano to macro 10 (2018) 145-158. [18]
T. Kashyap, K.K. Pramanik, N. Nath, P. Mishra, A.K. Singh, S. Nagini, A. Rana, R.
Mishra, Crosstalk between Raf-MEK-ERK and PI3K-Akt-GSK3beta signaling networks promotes chemoresistance, invasion/migration and stemness via expression of CD44 variants (v4 and v6) in oral cancer, Oral oncology 86 (2018) 234-243.
18
[19]
L. Chang, P.H. Graham, J. Ni, J. Hao, J. Bucci, P.J. Cozzi, Y. Li, Targeting
PI3K/Akt/mTOR signaling pathway in the treatment of prostate cancer radioresistance, Critical reviews in oncology/hematology 96 (2015) 507-517. [20]
C. Chen, S. Zhao, A. Karnad, J.W. Freeman, The biology and role of CD44 in cancer
progression: therapeutic implications, J Hematol Oncol 11 (2018) 64. [21]
J. Ni, P. Cozzi, J. Hao, W. Duan, P. Graham, J. Kearsley, Y. Li, Cancer stem cells in
prostate cancer chemoresistance, Current cancer drug targets 14 (2014) 225-240. [22]
S. Misra, S. Ghatak, B.P. Toole, Regulation of MDR1 expression and drug resistance
by a positive feedback loop involving hyaluronan, phosphoinositide 3-kinase, and ErbB2, The Journal of biological chemistry 280 (2005) 20310-20315. [23]
F. Ji, D. Ma, Z. Liu, X. Xie, Rosiglitazone amplifies the sensitivity of docetaxel and
reduces the expression of CD44v6, Oncology letters 7 (2014) 1284-1288. [24]
L. Chang, P.H. Graham, J. Hao, J. Ni, J. Bucci, P.J. Cozzi, J.H. Kearsley, Y. Li,
Acquisition of epithelial-mesenchymal transition and cancer stem cell phenotypes is associated with activation of the PI3K/Akt/mTOR pathway in prostate cancer radioresistance, Cell death & disease 4 (2013) e875. [25]
S.H. Choi, A.R. Kim, J.K. Nam, J.M. Kim, J.Y. Kim, H.R. Seo, H.J. Lee, J. Cho, Y.J.
Lee, Tumour-vasculature development via endothelial-to-mesenchymal transition after radiotherapy controls CD44v6(+) cancer cell and macrophage polarization, Nature communications 9 (2018) 5108. [26]
T. Liu, Q. Sun, Q. Li, H. Yang, Y. Zhang, R. Wang, X. Lin, D. Xiao, Y. Yuan, L.
Chen, W. Wang, Dual PI3K/mTOR inhibitors, GSK2126458 and PKI-587, suppress tumor progression and increase radiosensitivity in nasopharyngeal carcinoma, Molecular cancer therapeutics 14 (2015) 429-439.
19
[27]
L. Chang, P.H. Graham, J. Hao, J. Ni, J. Bucci, P.J. Cozzi, J.H. Kearsley, Y. Li,
PI3K/Akt/mTOR pathway inhibitors enhance radiosensitivity in radioresistant prostate cancer cells through inducing apoptosis, reducing autophagy, suppressing NHEJ and HR repair pathways, Cell death & disease 5 (2014) e1437. [28]
G. Wohlleben, K. Hauff, M. Gasser, A.M. Waaga-Gasser, T. Grimmig, M. Flentje, B.
Polat, Hypoxia induces differential expression patterns of osteopontin and CD44 in colorectal carcinoma, Oncol Rep 39 (2018) 442-448. [29]
B. Krishnamachary, M.F. Penet, S. Nimmagadda, Y. Mironchik, V. Raman, M.
Solaiyappan, G.L. Semenza, M.G. Pomper, Z.M. Bhujwalla, Hypoxia regulates CD44 and its variant isoforms through HIF-1alpha in triple negative breast cancer, PloS one 7 (2012) e44078. [30]
Y.Y. Xu, M. Guo, L.Q. Yang, F. Zhou, C. Yu, A. Wang, T.H. Pang, H.Y. Wu, X.P.
Zou, W.J. Zhang, L. Wang, G.F. Xu, Q. Huang, Regulation of CD44v6 expression in gastric carcinoma by the IL-6/STAT3 signaling pathway and its clinical significance, Oncotarget 8 (2017) 45848-45861. [31]
S. Damm, P. Koefinger, M. Stefan, C. Wels, G. Mehes, E. Richtig, H. Kerl, M. Otte,
H. Schaider, HGF-promoted motility in primary human melanocytes depends on CD44v6 regulated via NF-kappa B, Egr-1, and C/EBP-beta, The Journal of investigative dermatology 130 (2010) 1893-1903. [32]
Hao JL, Ni J, Graham P, Cozzi P, Bucci J, Kearsley J, L. Yi, The CD44 isoforms in
prostate cancer metastasis and progression, World Journal of Cancer Research 1 (2013) 3-14. [33]
M. Todaro, M. Gaggianesi, V. Catalano, A. Benfante, F. Iovino, M. Biffoni, T.
Apuzzo, I. Sperduti, S. Volpe, G. Cocorullo, G. Gulotta, F. Dieli, R. De Maria, G. Stassi, CD44v6 is a marker of constitutive and reprogrammed cancer stem cells driving colon cancer metastasis, Cell stem cell 14 (2014) 342-356.
20
[34]
S. Patel, B. Waghela, K. Shah, F. Vaidya, S. Mirza, S. Patel, C. Pathak, R. Rawal,
Silibinin, A Natural Blend In Polytherapy Formulation For Targeting Cd44v6 Expressing Colon Cancer Stem Cells, Scientific reports 8 (2018) 16985. [35]
Z. Wang, H. Sun, J. Provaznik, T. Hackert, M. Zoller, Pancreatic cancer-initiating cell
exosome message transfer into noncancer-initiating cells: the importance of CD44v6 in reprogramming, Journal of experimental & clinical cancer research : CR 38 (2019) 132. [36]
S. Misra, P. Heldin, V.C. Hascall, N.K. Karamanos, S.S. Skandalis, R.R. Markwald, S.
Ghatak, Hyaluronan-CD44 interactions as potential targets for cancer therapy, The FEBS journal 278 (2011) 1429-1443. [37]
M. Casucci, B. Nicolis di Robilant, L. Falcone, B. Camisa, M. Norelli, P. Genovese,
B. Gentner, F. Gullotta, M. Ponzoni, M. Bernardi, M. Marcatti, A. Saudemont, C. Bordignon, B. Savoldo, F. Ciceri, L. Naldini, G. Dotti, C. Bonini, A. Bondanza, CD44v6-targeted T cells mediate potent antitumor effects against acute myeloid leukemia and multiple myeloma, Blood 122 (2013) 3461-3472. [38]
V. Leuci, G.M. Casucci, G. Grignani, R. Rotolo, U. Rossotti, E. Vigna, L.
Gammaitoni, G. Mesiano, E. Fiorino, C. Donini, A. Pisacane, L.D. Ambrosio, Y. Pignochino, M. Aglietta, A. Bondanza, D. Sangiolo, CD44v6 as innovative sarcoma target for CARredirected CIK cells, Oncoimmunology 7 (2018) e1423167.
Figure Legends Fig. 1. Expression of CD44v6 in PC-3M-CD44v6-KD, PC-3M-CD44v6-scr and PC-3M cell lines. (A) WB is shown to confirm the KD of CD44v6 in PC-3M cell line. ß-tubulin was chosen as a loading control. (B) KD of CD44v6 is further confirmed by qRT-PCR. Results are expressed as Mean ± SD (n=3). ***P<0.001. KD, knock-down; scr, scrambled shRNA control; WB, Western blot
21
Fig. 2. Tumour growth of PC-3M-CD44v6-KD and PC-3M-CD44v6-scr xenografts in CaP s.c. and orthotopic mouse models. (A) Graph is shown for tumour growth curves of PC-3MCD44v6-KD and PC-3M-CD44v6-scr cells in the s.c. xenograft model, the weekly measurements of PC-3M-CD44v6-KD xenografts showed a significant reduced tumour growth compared to the PC-3M-CD44v6-scr group (n=9, mean±SD, P<0.05). (B) At the end of the experiment, tumour weight from PC-3M-CD44v6-KD group mice was significantly decreased compared to that from PC-3M-CD44v6-scr group mice in s.c. model (n=9, mean±SD, * indicates P<0.05). (C) Representative images of tumours harvested from s.c. xenografts are shown. (D) Graph is shown for growth curves of PC-3M-CD44v6-KD and PC3M-CD44v6-scr xenografts in the orthotopic xenograft model (n=9, mean±SD, P<0.05). (E) At the end of the experiment, tumour weight from PC-3M-CD44v6-KD group mice was significantly decreased compared to that from PC-3M-CD44v6-scr group mice in orthotopic model (n=9, mean±SD, *P<0.05). (F) Representative images of tumours from orthotopic xenografts are shown. KD, knock-down; scr, scrambled shRNA control.
Fig. 3. In vivo effects of chemo-/radiosensitivity of PC-3M-CD44v6-KD and PC-3MCD44v6-scr cell lines in s.c. animal models. (A) Tumour growth curves are shown for DTX treatment groups in PC-3M-CD44v6-KD and PC-3M-CD44v6-scr s.c. mouse models. (B) The ratios of DTX-treated versus VC-treated tumour volumes (DTX/VC) were calculated and plotted from the start of DTX treatment to the end of experiments in s.c. mouse model. (C) Tumour growth curves are shown for RT groups in PC-3M-CD44v6-KD and PC-3MCD44v6-scr s.c. mouse model. (D) The ratios of RT versus SC tumour volumes (RT/SC) were calculated and plotted from the start of RT to the end of experiments in s.c. mouse model. DTX, docetaxel; KD, knock-down; SC, sham control; RT, radiotherapy; scr, scrambled shRNA control; VC, vehicle control.
Fig. 4. EpCAM, PI3K/mTOR/Akt pathway proteins, Ki-67, CD31, Caspase-3 (active) and γH2AX expressions in s.c. CaP mouse xenografts. (A) Significantly reduced expression of CD44v6 was observed after KD of CD44v6 in the s.c. xenograft. (B-E) Phosphorylated PI3K/Akt/mTOR signalling proteins (p-mTOR and p-Akt) were downregulated in CD44v6KD group compared to CD44v6-scr group, whereas no obvious changes were seen in total mTOR and Akt between the two groups . (F) Strong expression of Ki-67 was observed in PC3M-CD44v6-scr s.c. xenografts while after KD of CD44v6, the expression of Ki-67 is weak. (G) Markedly reduced CD31 expression was seen in PC-3M-CD44v6-KD xenografts 22
compared to PC-3M-CD44v6-scr group after DTX treatment. (H) An increased level of active Caspase-3 expression was seen in PC-3M-CD44v6-KD xenografts compared to PC3M-CD44v6-scr xenografts after treatment with DTX. (I) After RT, an increased level of γH2AX expression was seen in PC-3M-CD44v6-KD xenografts compared to PC-3MCD44v6-scr xenografts. Magnification x400 in all images. DTX, docetaxel; KD, knock-down; RT, radiotherapy; s.c., subcutaneous; scr, scrambled shRNA control.
Fig. S1. Validation of CD44v6-KD using three independent shRNA in PC-3M cell line. WB is shown to confirm the KD of CD44v6 in PC-3M cell line using three independent shRNAs and one scrambled shRNA control. ß-tubulin was chosen as a loading control. No significant difference was observed. KD, knock-down; scr, scrambled shRNA control; shRNA, short hairpin RNA; WB, Western blot.
23
-3 M
-C
PC-3M
PC
PC-3M-CD44v6-KD PC-3M-CD44v6-scr
-C
β-tubulin
-3 M
CD44v6
D 44 v6 -
K
D
sc r
-3 M D 44 v6 -
PC
Normalised CD44v6 Expression (%)
B
PC
A
A
B
C
D
E
F
A
B
C
D
PC-3MPC-3MCD44V6-KD CD44v6-scr
PC-3MCD44V6-KD
PC-3MCD44v6-scr
A
CD44v6
F
Ki-67
B
Akt
G
CD31
C
p-Akt
H
Caspase-3 (active)
D
mTOR
I
γH2AX
E
p-mTOR
COI statement
We have no conflict of interest for this submission.
CD44 variant 6 is associated with prostate cancer growth and chemo/radiotherapy response in vivo Jie Nia,b, Belamy B. Cheungc,d, Julia Beretova,b,e, Wei Duanf, Joseph Buccia,b, David Maloufa,g, Peter Grahama,b, Yong Lia,b,h,*
a
Cancer Care Centre, St George Hospital, Kogarah, NSW 2217, Australia
b
St George and Sutherland Clinical School, Faculty of Medicine, UNSW Sydney, NSW 2052,
Australia c
Children's Cancer Institute Australia, Lowy Cancer Research Centre, UNSW Sydney, NSW
2052, Australia d
School of Women's & Children's Health, Faculty of Medicine, UNSW Sydney, NSW 2052,
Australia e
Anatomical Pathology, NSW Health Pathology, St George Hospital, Kogarah, NSW 2217,
Australia f
School of Medicine and Centre for Molecular and Medical Research, Deakin University, Waurn
Ponds, VIC 3216, Australia g
Department of Urology, St George Hospital, Kogarah NSW 2217, Australia
h
School of Basic Medical Sciences, Zhengzhou University, Henan 450001, China
*Correspondence Yong Li, Cancer Care Centre, St George Hospital, Level 2, 4-10 South St, Kogarah NSW 2217 Australia. Tel: +61-2-91131338; Fax: +61-2-91131386; Email: [email protected]