Sonic hedgehog signaling is associated with resistance to zoledronic acid in CD133high/CD44high prostate cancer stem cells

Eda Acikgoz1 · Gunel Mukhtarova2,3 · Araz Alpay4 · Cigir Biray Avci5 · Bakiye Goker Bagca5 · Gulperi Oktem6,7


Cancer stem cells (CSCs) are a unique population that has been linked to drug resistance and metastasis and recurrence of prostate cancer. The sonic hedgehog (SHH) signal regulates stem cells in normal prostate epithelium by affecting cell behavior, survival, proliferation, and maintenance. Aberrant SHH pathway activation leads to an unsuitable expansion of stem cell lineages in the prostate epithelium and the transformation of prostate CSCs (PCSCs). Zoledronic acid (ZOL), one of the third-generation bisphosphonates, effectively prevented bone metastasis and treated advanced prostate cancer despite androgen deprivation therapy. Despite strong evidence for the involvement of the SHH in human PCSCs survival and drug resistance, the roles of SHH in the PCSCs-related resistance to ZOL remain to be fully elucidated. The present study aimed to investigate the role of the SHH pathway in ZOL resistance of PCSCs in 2D and three 3D cell culture conditions. For this purpose, we isolated CD133high/ CD44high PCSCs using a flow cytometer. Following ZOL treatment, mRNA and protein expressions of the components of the SHH signaling pathway in PCSCs and non-CSCs were analyzed using qRT-PCR and Immunofluorescence staining, respectively. Our finding suggested that SHH signaling may be activated by different mecha- nisms that lead to avoidance of the inhibition effect of ZOL. Thereby, SHH pathways may be associated with the resistance to ZOL developed by prostate CSCs. Inhibition of CSCs-related SHH signaling along with ZOL treatment should be con- sidered to achieve improvement in survival or delayed treatment failure and prevention of the CSCs-related drug resistance.

Keywords Cancer stem cell · Sonic hedgehog · Zoledronic acid · Drug resistance · Three-dimensional culture


Prostate cancer (PCa) remains the most common can- cer and second cancer-related malignancy in men [1]. Despite responding to radiotherapy, chemotherapy, or androgen-deprivation therapy (ADT) initially, resistance to treatment develops, and subsequently, the progression of disease and metastasis occurs in practically all PCa patients [2]. Although significant advances have been made in the diagnosis and treatment of PCa, recurrence of prostate scan- cer after treatment poses a clinically significant problem. In this context, elucidating the cellular population and basic molecular mechanisms governing the progression of PCa will contribute to the development of effective treatment strategies.
As with other types of cancer, PCa consists of heteroge- neous cells, and a small population within cancer cells is responsible for tumor formation, progression, invasion, drug resistance, and metastasis [3, 4]. Cells exhibiting these spe- cific properties are referred to as cancer stem cells (CSCs).
There is a strong consensus on targeting and eliminating cancer stem cells for effective treatment in PCa treatment. To identify and isolate CSCs, commonly used CD133 and CD44 cell surface markers are widely used. Both CD44 and CD133 markers are glycoproteins and found to be associated with stemness properties [5, 6]. CD133 and CD44 positive cells are considered as prostate cancer stem cells (PCSCs) and are commonly used in the CSCs-related drug resistance studies [7, 8].
The Food and Drug Administration approved zoledronic acid (ZOL) for the prevention or reduction of osteoclastic bone resorption and skeletal morbidity in breast and prostate cancers [9]. Farnesyl diphosphate synthase (FDPS), which is the mevalonate pathway enzyme expressed at high levels in prostate cancer, plays an essential role in prostate cancer development and aggression [10]. By binding and inhibiting FDPS, ZOL prevents the production of cholesterol, other sterols, and key enzymes such as farnesyl pyrophosphate and geranyl–geranyl pyrophosphosphate [10]. There is an increasing experimental evidence that ZOL has anti-tumor and anti-metastatic effects; inhibition of proliferation of tumor cells, induction of apoptosis, and prevents invasion of cancer cells [11, 12]. Although it is the most widely used bisphosphonate for preventing or reducing osteoclastic skel- etal-related complications, the insufficiency identified for ZOL in terms of improving survival or delayed treatment failure in PCa patients has been suggested may be related to the development of drug resistance against ZOL [13–16]. In a study by Mileno et al., It was reported that ZOL resist- ance of PCa cells might be associated with p38-MAP kinase activation [17]. Therefore, understanding the mechanisms underlying ZOL resistance of PCa cells is essential for the development of new therapeutic strategies against drug resistance.
Sonic Hedgehog (SHH) is an evolutionarily conserved embryonic signaling pathway that regulates patterned growth in different tissues [18]. Mostly inactive or par- tially active SHH pathway ensures the maintenance of pluripotent cells and somatic stem cells and is essential for the regeneration and tissue repair in the different tis- sue such as the lung, brain, internal organs, and prostate in the adult organism. Thereby mutation and/or epigenetic alterations in SHH members result in the deregulation and aberrant activation of this pathway and give rise to SHH-dependent tumors [19]. Prostate tissue homeostasis, growth, cell polarity, and proliferation are controlled by SHH pathway activation in normal prostate growth and development [20]. Increasing evidence suggests that drug resistance of cancer cells is associated with the SHH path- way [21, 22]. SHH signaling pathway regulates stem cells like cell behavior, survival, proliferation, and maintenance in normal prostate epithelium. Aberrant SHH pathway activation can cause an unsuitable expansion of stem cell lineages in the prostate epithelium and the transformation of PCSCs. The development of clinical trials that target the SHH pathway may lead to more effective treatment such as decreasing multidrug resistance and inhibition of tumor growth by the elimination of PCSCs. Knowing that one of the key CSCs–related pathways SHH plays a critical role in human PCSCs survival and drug resistance, we investi- gated the effects of ZOL on the SHH pathway in PCSCs in two dimensional (2D) and three dimensional (3D) culture systems. We found that SHH pathways may be associated with ZOL-induced anti-cancer activity. Combination ther- apy to inhibit SHH signaling along with ZOL treatment may be considered to achieve improvements in survival or delayed treatment failure and prevention of CSCs-related drug resistance.

Materials and methods

Cell cultures and reagents

PC-3 and DU-145 were purchased from American Type Culture Collection (Manassas, VA, USA) and were grown in RPMI 1640 (Lonza, Basel, Switzerland) culture medium containing 10% heat-inactivated fetal bovine serum (Gibco, Invitrogen Life Technologies, Paisley, UK), 1% penicillin and streptomycin (Sigma-Aldrich, St Louis, MO, USA) respectively. Antibodies used were anti-Smo (1:100 diluted; Santa Cruz Biotechnology, USA, sc- 13943), anti-Patched 1 (1:100 diluted; Santa Cruz Biotechnology, USA, sc- 9016), anti-SHH (1:100 diluted; Santa Cruz Biotechnology, USA, sc-9024), goat anti-rabbit immunoglobulin G-fluorescein isothiocyanate (FITC) (1:100 diluted; sc-2012, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Zoledronic acid was provided by Sigma-Aldrich (Darmstadt, Germany) and was prepared as a 5 mM stock solution in a sodium hydrox- ide (NaOH).

Fluorescence‑activated cell sorting (FACS)

Cell surface markers are used to isolate stem cells in can- cer cells with a heterogeneous cell population. CSCs in the prostate cancer cell lines (DU-145 and PC-3) were isolated using a combination of CD133 and CD44 surface markers as previously described [16] using a FACSAria flow cytom- eter. Briefly; 1 × 106 cells were incubated with an antibody (diluted 1:100 in FACS wash with 0.5% bovine serum albu- min; 2 mM NaN3 and 5 mM EDTA) for 15 min at 4 °C. Prostate cancer cells were labeled with PE-labeled CD133/1 (clone AC133/1; Miltenyi Biotec Ltd.) and FITC-labeled CD44 (clone G44-26; BD Biosciences). Then, prostate CSCs were sorted as CD133high/CD44high population.

Determination of half‑maximal inhibitory concentrations (IC50) of ZOL

Muse Count and Viability kit was used to evaluate the effect of ZOL on cell viability. Our group has previously determined IC50 values of ZOL for DU-145 CSCs and non-CSCs [12]. In this study, doses obtained from the previous study were used for DU-145 CSCs and non-CSCs cells. PC-3 CSCs and PC-3 non-CSCs cells were seeded and cultured at a density of 1 × 104 cells/well in 6-well plates (Becton Dickinson, San Jose, CA) in triplicate. ZOL was added at the increasing doses (0 µM,10 µM, 25 µM, 50 µM, 100 µM, 150 µM) and cells were incubated at 37 °C in 5% CO2 for 24 h, 48 h, and 72 h. After incubation, all cells were collected and diluted with phosphate- buffered saline (PBS). Cell viability was analyzed using the Muse Count and Viability Kit (MuseTMCell Analyzer; Mil- lipore, Billerica, MA, USA) and MUSE Cell Analyzer, accord- ing to the manufacturer’s instructions. Data are presented as proportional viability (%) by comparing the treated group with the untreated cells (control), the viability of which was assumed to be 100%.

Spheroid formation assay and drug treatment

For spheroid formation, 1.5% agar-coated 6-well plates were used. DU-145 CSCs and PC-3 CSCs isolated according to CD133high and CD44high surface properties were seeded at a density of 1 × 104 cells/mL per well in serum-free RPMI 1640 medium. Then, the plates were placed in the shaking incubator for approximately 1 h to allow the distribution of the cells. After the formation of the spheroids, DU-145 and PC-3 CSCs spheroids were treated with 108 μM and 150 μM ZOL, respectively for 72 h. The changes of spheroid num- bers and diameters were evaluated with representative images were taken under an inverted microscope equipped with a camera (Olympus BX51, Hamburg, Germany) at 4X and 10X magnifications, respectively. All spheroids in each well were counted to measure the number of spheroids. In evaluating the changes in spheroid diameter, 10 different spheroids were photographed in randomly selected areas in each well, and diameters were measured. The diameter of each spheroid was measured using ImageJ (NIH, USA). The spheroids volumes were calculated from major (L) and minor (W) axes using the following formula: 0.5 *L * W2. All experiments were carried out with three replicates, and the results were blindly measured and counted by two independent researchers.

Determination of expressional changes of SHH, GLI1, SMO, PTCH1 genes by RT‑PCR after ZOL treatment

To detect gene expression of SHH, GLI1, SMO, PTCH1 in the experimental groups in total we use RT2 qPCR Primer Assay for Human SHH (cat. no: PPH02405A), RT2 qPCR Primer Assay for Human SMO (cat. no: PPH02222C), RT2 qPCR Primer Assay for Human PTCH1 (cat. no: PPH00225C), RT2 qPCR Primer Assay for Human GLI1 (cat. no: PPH00153A). Total RNA was extracted from) ZOL-treated and untreated both DU-145 CSCs and PC- 3CSCs monolayers and spheroids, and non-CSCs using RNeasy Plus Mini Kit (Qiagen) and this was followed by cDNA synthesis. The expression of these were analyzed by LightCycler® 480 real-time PCR. Data analysis was per- formed using SABioscience’s proprietary online program. All qPCR gene expression was normalized to the expres- sion of GAPDH [23] and analyzed using the ΔΔCt method. Fold change of genes expressions were calculated using 2^(- ΔΔCt) formula. The p-values are calculated based on a Student’s t-test of the replicate 2^(- Delta CT) values for each gene in the control group and other groups. If the p-value in the two-tailed test was lower than 0.05 and the fold change was lower or greater twofold, then the changes were considered significantly down-regulated or up-regu- lated, respectively. If the p-value in the two-tailed test was higher than or equal to 0.05, then the changes were accepted as not significant.

Immunofluorescence staining

DU-145 CSCs, DU-145 non-CSCs, PC-3 CSCs, PC-3 non-CSCs cells were cultured. 1 × 104 cells were plated on lysine coated coverslips and fixed in 4% paraformalde- hyde for 15 min. For permeabilization, the cells were kept in 1% Triton X-100 for 10 min at room temperature. After washing three times with PBS, the cells were blocked with PBS containing 5% bovine serum albumin for 1 h. Follow- ing incubation with antibodies overnight at 4 °C, the cells were treated with secondary antibody (1:100 dilution) for 1 h at room temperature. The immunostained cells after mounted in a mounting medium (sc-24941, UltraCruz mounting medium) containing DAPI, the cells were visual- ized using a fluorescence microscope (Olympus BX-51 and the Olympus C-5050 digital test). The fluorescent images were then analyzed by the ImageJ (Image analysis software, National Institutes of Health, Bethesda, MD). Firstly, all the images were converted into channels in grayscale 8‐bit by the “split channels command”. Then “area’’, “integrated intensity’’ and ‘’mean gray value’’ were selected from the analyze menu. We measured the area, integrated density, and mean gray value by selecting one cell and 3 + selections from around the selected cell as the background we meas- ured the area, integrated density and mean gray value. The calculation for corrected total cell fluorescence (CTCF) was calculated using CTCF = integrated density–(area of selected cell × mean fluorescence of background readings) formula. Obtained CTCF values of ZOL-treated cells were then equalized against the mean CTCF of untreated cells to determine fold changes.

Statistical analyses

All experiments were carried out at least three times. The data were expressed as ± standard deviation (SD) and were analyzed by univariate analysis of variance and independent simple test. Results were analyzed by IBM SPSS Statistics for Windows, Version 25.0. (Released 2017, Armonk, NY: IBM Corp.). After ZOL treatment, the variables obtained in relation to the number and diameters of spheroids were analyzed by the factorial ANOVA method. The error esti- mates obtained from the analysis were checked for normal- ity. Kolmogorov Smirnov methods were used for diameter variables and Shapiro Wilk methods were used for numerical variables. Variables with significant interaction of time x concentration were evaluated using the Independent Two Group Student-t Test. Statistically significant differences were considered if p-values less than 0.05 (p < 0.05). Results Purity and sorting rates of CD 133high/ CD44high sorted and non‑sorted subpopulations Prostate CSCs were isolated as CD133high/CD44high popu- lation from DU-145 and PC-3 human prostate cancer cells by FACS (Fig. 1a and b). The rates of sorted cells (CSCs) and non-sorting cells (non-CSCs) were 3.4 ± 5.4% and 96.6 ± 5.4% (Fig. 1a) in DU-145, and 4.8 ± 5.4 and 96.2 ± 5.4 in PC-3 (Fig. 1b), respectively. A post-sort analysis was per- formed to detect the purity of the CSCs populations and was determined to be >89%.

Increasing cytotoxicity of CD133high/CD44high prostate CSCs with ZOL

To determine IC50 concentration of ZOL on PC-3 CSCs and non-CSCs subpopulation, the cells were treated with ZOL at the increasing doses (0 µM, 10 µM, 25 µM, 50 µM, 100 µM, 150 µM) were incubated at 37 °C in a 5% CO2 for 24 h, 48 h, and 72 h. Cell viability was measured using the Muse Cell Analyzer. The effects of ZOL on PC-3 CSCs and PC-3 non- CSCs cell viability in a time- and concentration-dependent manner were shown in Figs. 2 and 3, respectively. Though the IC50 concentration of ZOL could not be obtained for 24 h and 48 h, it was determined as 150 μM and 111,7 μM in 72 h, respectively, for PC-3 CSCs and PC-3 non-CSCs (Fig. 3). IC50 values of ZOL for DU-145 CSCs and non- CSCs, previously determined by our group, were 108 μM and 96 μM in 72 h, respectively [12]. IC50 concentration between CSCs and non-CSCs in both PC-3 and DU-145 cells were significantly different.

Effects of ZOL on the numbers and diameters of spheroids

3D cell culture systems reflect cell–cell contacts and physi- cal situations better than 2D culture systems. Notably, the sensitivity and/or resistance to the drugs in the 3D system may differ from two-dimensional systems. Therefore, we aimed to examine the effects of ZOL on the spheroidal structures of DU-145 CSCs and PC-3 CSCs. DU-145 and PC-3 CSCs were cultured on agar-coated 6-well plates for 3 days (DU-145 CSCs) or 5 days (PC-3 CSCs), followed by treatment with 108 μM and 150 μM ZOL, respectively for 72 h. In the evaluation of prostatospheres formed by PC3 and DU-145 cancer stem cells in three-dimensional cul- ture systems, criteria such as packing the cells (loose and/ or compact), the numbers, and diameters of spheroids were considered. The first observations about the drug-free con- trol groups, the time of compact spheroid formation, and the morphology of the spheroidal structures were different in both cell lines. As shown in Fig. 4, DU-145 CSCs formed morphologically more diffuse and irregular spheroidal struc- tures. In contrast, PC-3 CSCs exhibited a distinctly rounded and better characterized spheroidal structure. After 72 h, the diameters of 3D spheroids of PC-3 CSCs were consider- ably higher than DU-145 CSCs. We further investigated the responses of the spheroids to a drug, aiming to determine whether ZOL affects DU-145 CSCs and PC-3 CSCs in a 3D context. No significant changes in spheroid numbers and diameters were detected in both cell lines after ZOL administration (p > 0,05). Based on these results, it can be concluded that the cells cultured in the 3D system are more resistant to ZOL than cells cultured in 2D system (Fig. 4).

Effect of ZOL acid on the expression of the SHH pathway components in both prostate CSCs monolayers and spheroids

To investigate ZOL effects on the SHH pathway, we exam- ined expression changes of SHH pathway genes SHH, SMO, GLI1, and PTCH1 by RT-PCR in PC-3 and DU-145 CSCs (cultured in 2D and 3D culture system) and 108 μM and 96 μM ZOL (DU-145 CSCs and non-CSCS, respectively) for 72 h. Fold change of genes expressions were calcu- lated using 2^(- ΔΔCt) formula. Fold change values less or greater than 2 (twofold) genes were considered signifi- cantly expressed. GLI1 was up-regulated 6,36 (p < 0,05) and 4,06 times (p < 0,05) in both DU-145 CSCs monolayers and spheroids, respectively. SHH expression was also 3,39 (p < 0,05) and 2,11 (p < 0,05) times higher in ZOL-treated DU-145 CSCs monolayers and spheroids, respectively, compared to controls. SMO and PTCH1 were up-regulated 2,50 (p < 0,05) and 2,08 (p < 0,05) times, respectively in DU-145 CSCs spheroids but not significantly changed in ZOL-treated DU-145 CSCs monolayers than untreated DU-145 CSCs. However, mRNA expression of SMO was−2,99 (p < 0,05) times lower and PTCH1 was not signifi- cantly changed in DU-145 non-CSCs after ZOL treatment (Table 1). Expressional changes were different in PC-3 from DU-145. GLI1, PTCH1, and SHH genes were down-regu- lated −2,13 (p < 0,05), −2,35 (p < 0,05) and −2,35 (p < 0,05) times, respectively in PC-3 CSCs monolayer whereas all of the genes (GLI1, PTCH1, SHH, and SMO) were not signifi- cantly changed in PC-3 spheroids after ZOL treatment. All genes (GLI1, SHH, PTCH1 and SMO) were also not signifi- cantly changed in ZOL-treated PC-3 non-CSCs compared to controls (Table 2). SHH, SMO, and PTCH1 protein expression in prostate CSCs and non‑CSCs after ZOL treatment To investigate protein expression and localization of PTCH1, SHH, SMO after ZOL-treated cells, following treatment with 150 μM and 111,7 μM ZOL (PC-3 CSCs and non- CSCs, respectively) and 108 μM and 96 μM ZOL (DU- 145 CSCs and non-CSCS, respectively) for 72 h, immu- nostaining of these proteins was carried out. There are no significant changes we determined in the expression of the PTCH1 receptor in both DU-145 and PC-3 CSCs and non- CSCs. SHH was down-regulated in PC-3 CSCs and non changed in PC-3 non-CSCS, whereas it was up-regulated in both DU-145 CSCs and non-CSCs. SMO was significantly down-regulated in both DU-145 and PC-3 non-CSCs but did not change in both PC-3 and DU-145 CSCs (Fig. 5). Discussion The presence of treatment-resistant CSCs in the tumor mass is one of the most important obstacles to cancer treatment success. CSCs, which are resistant to chemotherapy and radiotherapy compared to the bulk population, play impor- tant roles in tumor relapse after anti-cancer treatment. There- fore, the development of anti-cancer therapeutics targeting CSCs and elucidating the mechanisms underlying drug resistance may direct the improvement of treatment strate- gies. Signaling pathways in embryonic development are one of the important building blocks of the architecture which creates resistance to chemotherapeutic agents in cancer stem cells, which are expressed as the “seed of the tumor” [24]. At this point, the “drug resistance-signaling pathway” axis may be one of the important steps in the era of new oncological treatments associated with drug resistance. ZOL is the most widely used bisphosphonate which prevents disease-related skeletal complications in PCa patients. ZOL also inhibited prostate cancer cell proliferation and growth by inducing apoptosis and autophagy in vitro [12, 25]. However, the improvement effect of ZOL treatment on survival and pre- ventive effect on metastatic progression and cancer-associ- ated death have been found insufficient in PCa patients [13]. In this study, we aimed to demonstrate the effects of ZOL on PCSCs in both 2D and 3D cell culture systems and investi- gated the possible relationship of the ZOL and SHH pathway to reveal the mechanisms underlying ZOL-resistance. PCa is composed of genetically- and phenotypically- distinct tumorigenic cells which can be divided into bulk cancer cells and small population stem or stem-like cells that have self-renewal capacity. It was suggested that one of the main causes of the insufficiency of current thera- peutic approaches that target bulk cancer cells (non-CSCs) is drug resistance developed by PCSCs [26]. ZOL is the most widely used bisphosphonate which prevents disease- related skeletal complications in PCa patients [9]. ZOL also inhibits prostate cancer cell proliferation and growth by inducing apoptosis and autophagy in vitro [12, 25, 27]. However, the improving effect of ZOL treatment on survival and preventive effect on metastatic progression and cancer-associated death were found to be insufficient in PCa patients [13, 14, 28, 29]. In previous studies, the effects of ZOL on CSCs with metastatic properties were reported [3, 12, 30]. Buhler et al. demonstrated that ZOL inhibits motility and velocity of breast cancer stem-like progenitor cells [30]. Zoledronic acid exhibits a similar inhibiting effect on the cervical CSCs and attenuates their stemness phenotype [31]. In our study, we examined the effects of ZOL on both bulk and cancer stem cell popula- tions. Our results showed that ZOL has cytotoxic effects on PCSCs at high doses (compared to bulk population), especially in 2D culture systems. However, CSCs were found to be more resistant to ZOL treatment in both cell lines. Also, it has been shown that DU-145 CSCs by over- expression of a cluster of anti-apoptotic genes developed resistance to ZOL in the 2D culture system [12]. Addition- ally, M. R. Milone et al. demonstrated that ZOL-resistant DU-145R80 prostate cancer cells increasingly expressed anti-apoptotic proteins and acquire CSCs features as well in the 2D culture system [17]. We found that as well as DU-145 CSCs, PC-3 CSCs were more resistant to ZOL treatment than non-CSCs (IC50 were108 μM and 150 μM, respectively, for 72 h). Induction of cytotoxicity by ZOL in PC-CSCs was the dose-and time-dependent manner in vitro. In our study, both CD 133high/ CD44high PC-3 and DU-145 CSCs showed possess spherogenic potential in a non-adherent culture. We have determined that the effects of ZOL on 2D and 3D culture systems have differ- ent results. In 3D systems, spheroidal structures are more resistant to ZOL treatment. Additional to the conventional two-dimensional (2D) monolayers CSCs, tree-dimensional (3D) CSCs tend to form spheroidal aggregates. Sphere formation ability is considered one of the characteristic features of CSCs and has been shown linked to increased drug resistance. Compared to conventional two- dimensional (2D) monolayers, CSCs tend to form spheroid aggregates in three-dimensional culture systems, similar to in vivo models. In 3D culture systems, cancer cells exhibit different responses to drugs than cells cultured in 2D for a number of reasons, and cancer cells are more sensitive to drugs in 2D culture systems [32]. Previous studies have shown that cell polarity, cell–cell contacts and interactions, tumor geometry, and the microenvironment of the spheroidal structure can significantly affect the response of the drug [33, 34]. It was determined that PCSCs display different responses to ZOL in 2D and 3D culture systems. Our results showed that spheroid structures were more resistant to ZOL treatment. Spheroid structures formed by cancer cells exhibit different properties in terms of morphological, biophysical, and molecular aspects, and these properties cause changes in response to chemotherapeutic drugs [35]. In a study by Reidl et al., the signal pathways were revealed to be rearranged in spheroid structures formed in 3D culture systems and this changed the response of cancer cells to the drug [36]. Drug resistance is a major complication and persistent problem in cancer therapy and to overcome these problems and ensure successful treatment, drug resistance-related pathways in the spheroid structures formed by CSCs need to be investigated. PCSCs share common phenotypic and functional charac- teristics and stem cell-related pathways such as SHH, Wnt, Notch, and BMP with normal prostate stem cells [37]. Addi- tional to the tissue homeostasis and cell polarity action of SHH pathway in normal tissues, abnormal activity of this pathway is caused by the development of prostate cancer from an inappropriate expansion of prostatic epithelial stem cell lineages [20]. It was shown that increased activation of the SHH pathway is closely related to the high risk, aggres- siveness, tissue invasion, and increased metastasis potential in advanced and androgen resistant PCa [18, 38]. Two- and three-dimensional in vitro cell culture studies showed that the SHH signaling pathway controls growth, development, and survival of cancer cells, also leads to increased cancer cell proliferation in various cancer types including prostate, colon, and ovarian cancer [20, 39, 40]. The SHH pathway also regulated proliferation, self-renewal capacity, multi- drug resistance of PCSCs by increasing GLI-1- mediated transcription of target genes such as the ABC transporter proteins and also Nanog, Oct4, Sox2, and c-Myc [41]. In our study, we found that the SHH pathway is expressed at high levels in PCSC, which are closely associated with drug resistance, as well as in the bulk population. In this way, the SHH pathway involved in the epithelial-mesenchymal transition (EMT), survival, proliferation, stem cell func- tion of the prostate CSCs and plays an important role in the CSCs-related metastasis and drug resistance of prostate cancer [42, 43]. DU-145 and PC-3 prostate cancer cell lines, which are androgen-independent and derived from brain and bone metastasis respectively, are mostly used for prostate cancer studies that focus on the investigation of obstacles and molecular mechanisms underlying drug resistance. Both DU-145 and PC-3 harbor an active SHH pathway which is required for the proliferation of these cells [20, 44]. Two important components of the SHH signal pathway SHH and SMO, required cholesterol for their activation. Both mol- ecules are cholesterylated or cholesterol-modified proteins. Cholesterol is required for the maturation, releasing of SHH ligand, and its interaction with receptor PTCH1. SMO, also activated directly and indirectly by cholesterol [45–47]. Thereby, low levels and/or chemical activity of intracellular cholesterol leads to impairments SHH signaling pathway. ZOL modulates the mevalonate pathway and inhibited cholesterol biosynthesis [48, 49]. Thereby ZOL may main- tain low levels of intracellular cholesterol. In this way, ZOL may impair the maturation and activation of SHH and SMO and can lead to the inhibition of the SHH signaling pathway as well. On the other hand, PTCH1 maintains cholesterol at a low concentration and thereby prevents enrichment of SMO at the plasma membrane. After the interaction of SHH ligand with PTCH1 receptor, cholesterol concentration and cholesterol accessibility increase at the plasma membrane [50]. ZOL inhibits the synthesis of cholesterol [48] and also affects the activation of the SHH signaling pathway. Lee H. S., et al., showed that ZOL inhibits the CSCs-related SHH pathway and via targeting CSCs, ZOL inhibits cancer growth in HNSCC [51]. Despite the results of this study, we found that SHH-related genes were upregulated in both DU-145 CSC monolayers and spheroids after ZOL treatment. Our results showed that expression levels of the GLI, PTCH1 and SHH genes were increased in DU 145 CSCs after ZOL treatment. In the studies by Milone et al., it was reported that some signaling pathways related to aggressive and inva- sive phenotypes are activated in ZOL-resistant DU145R80 prostate cancer cells compared to parental DU145 cells [17, 51]. In another study by the same team, it was revealed that DU145R80 cells express cancer stem cell-like genes such as CD44, CD133, Nanog, Snail, Oct4 and ALDH at high levels, and ZOL-resistance may be associated with annexin A1 [52]. Unlike DU-145 CSCs, ZOL inhibited the CSC- related SHH pathway in PC-3 CSCs monolayers. But we did not found similar results in ZOL-treated PC-3 CSCs sphe- roids. No significant changes were detected in expressions of SHH components in PC3 CSCs. This suggested that in PC-3 and DU-145 CSCs, SHH signaling can be activated by diverse mechanisms that depend on the culture conditions and affected by ZOL differently. Our finding suggested that SHH signaling may be activated by different mechanisms that allow avoidance of the inhibition effect of ZOL. Thereby, SHH pathways may be associated with the resistance developed against ZOL. Combination therapy to inhibit SHH signaling along with ZOL treatment should be considered to achieve improve- ment on survival or delayed treatment failure and preven- tion of the CSCs-related drug resistance. References 1. Siegel RL, Miller KD, Jemal A (2015) Cancer statistics, 2015. CA Cancer J Clin 65:5–29. https://doi.org/10.3322/caac.21254 2. Merseburger AS, Bellmunt J, Jenkins C et al (2013) Perspectives on treatment of metastatic castration-resistant prostate cancer. Oncologist 18:558–567. https://doi.org/10.1634/theoncologist. 2012-0478 3. Wang G, Wang Z, Sarkar FH, Wei W (2012) Targeting prostate cancer stem cells for cancer therapy. Discov Med 13:135–142 4. Mei W, Lin X, Kapoor A et al (2019) The contributions of pros- tate cancer stem cells in prostate cancer initiation and metastasis. Cancers (Basel) 11:434. https://doi.org/10.3390/cancers11040434 5. Liou G-Y (2019) CD133 as a regulator of cancer metastasis through the cancer stem cells. Int J Biochem Cell Biol 106:1–7. https://doi.org/10.1016/j.biocel.2018.10.013 6. Marker PC (2007) Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells: Patrawala L, Calhoun T, Schneider- Broussard R, Li H, Bhatia B, Tang S, Reilly JG, Chandra D, Zhou J, Claypool K, Coghlan L. Urol Oncol Semin Orig Investig 25:277–278. https://doi.org/10.1016/j.urolonc.2007.02.003 7. Acikgoz E, Guven U, Duzagac F et al (2015) Enhanced G2/M arrest, caspase related apoptosis and reduced E-cadherin depend- ent intercellular adhesion by trabectedin in prostate cancer stem cells. PLoS One 10:1–17. https://doi.org/10.1371/journal.pone. 0141090 8. Erdogan S, Turkekul K, Dibirdik I et al (2018) Midkine down- regulation increases the efficacy of quercetin on prostate cancer stem cell survival and migration through PI3K/AKT and MAPK/ ERK pathway. Biomed Pharmacother 107:793–805. https://doi. org/10.1016/j.biopha.2018.08.061 9. Perry CM, Figgitt DP (2004) Zoledronic acid. Drugs 64:1197– 1211. https://doi.org/10.2165/00003495-200464110-00004 10. Seshacharyulu P, Rachagani S, Muniyan S et al (2019) FDPS cooperates with PTEN loss to promote prostate cancer progres- sion through modulation of small GTPases/AKT axis. Oncogene 38:5265–5280. https://doi.org/10.1038/s41388-019-0791-9 11. Wilson C, Ottewell P, Coleman RE, Holen I (2015) The dif- ferential anti-tumour effects of zoledronic acid in breast cancer– evidence for a role of the activin signaling pathway. BMC Can- cer 15:55. https://doi.org/10.1186/s12885-015-1066-7 12. Rouhrazi H, Turgan N, Oktem G (2018) Zoledronic acid over- comes chemoresistance by sensitizing cancer stem cells to apop- tosis. Biotech Histochem 93:77–88. https://doi.org/10.1080/10520 295.2017.1387286 13. James ND, Sydes MR, Clarke NW et al (2016) Addition of doc- etaxel, zoledronic acid, or both to first-line long-term hormone therapy in prostate cancer (STAMPEDE): survival results from an adaptive, multiarm, multistage, platform randomised controlled trial. Lancet 387:1163–1177. https://doi.org/10.1016/S0140- 6736(15)01037-5 14. Vale CL, Burdett S, Rydzewska LHM et al (2016) Addition of docetaxel or bisphosphonates to standard of care in men with localised or metastatic, hormone-sensitive prostate cancer: a sys- tematic review and meta-analyses of aggregate data. Lancet Oncol 17:243–256. https://doi.org/10.1016/S1470-2045(15)00489-1 15. Morii T, Ohtsuka K, Ohnishi H et al (2010) Inhibition of heat- shock protein 27 expression eliminates drug resistance of osteo- sarcoma to zoledronic acid. Anticancer Res 30:3565–3571 16. Kars MD, Işeri ÖD, Ural AU, Gündüz U (2007) In vitro evaluation of zoledronic acid resistance developed in MCF-7 cells. Antican- cer Res 27:4031–4037 17. Milone MR, Pucci B, Bruzzese F et al (2013) Acquired resist- ance to zoledronic acid and the parallel acquisition of an aggres- sive phenotype are mediated by p38-MAP kinase activation in prostate cancer cells. Cell Death Dis 4:e641–e641. https://doi. org/10.1038/cddis.2013.165 18. Skoda AM, Simovic D, Karin V et al (2018) The role of the Hedgehog signaling pathway in cancer: a comprehensive review. Bosn J basic Med Sci 18:8–20. https://doi.org/10.17305/bjbms. 2018.2756 19. Niyaz M, Khan MS, Mudassar S (2019) Hedgehog signaling: an achilles’ heel in cancer. Transl Oncol 12:1334–1344. https:// doi.org/10.1016/j.tranon.2019.07.004 20. Sanchez P, Hernández AM, Stecca B et al (2004) Inhibition of prostate cancer proliferation by interference with SONIC HEDGEHOG-GLI1 signaling. Proc Natl Acad Sci U S A 101:12561–12566. https://doi.org/10.1073/pnas.0404956101 21. Park SH, Jo MJ, Kim BR et al (2019) Sonic hedgehog pathway activation is associated with cetuximab resistance and EPHB3 receptor induction in colorectal cancer. Theranostics 9:2235– 2251. https://doi.org/10.7150/thno.30678 22. Sims-Mourtada J, Izzo JG, Ajani J, Chao KSC (2007) Sonic Hedgehog promotes multiple drug resistance by regulation of drug transport. Oncogene 26:5674–5679. https://doi.org/10. 1038/sj.onc.1210356 23. Di Vito A, Chiarella E, Baudi F et al (2020) Dose-depend- ent effects of zoledronic acid on human periodontal liga- ment stem cells: an in vitro pilot study. Cell Transplant 29:963689720948497–963689720948497. https://doi.org/10.1177/0963689720948497 24. Clara JA, Monge C, Yang Y, Takebe N (2020) Targeting sig- nalling pathways and the immune microenvironment of cancer stem cells — a clinical update. Nat Rev Clin Oncol 17:204–232. https://doi.org/10.1038/s41571-019-0293-2 25. Ji-Fan L, Yi-Chia L, Yi-Hsuan L et al (2011) Zoledronic acid induces autophagic cell death in human prostate cancer cells. J Urol 185:1490–1496. https://doi.org/10.1016/j.juro.2010.11. 045 26. Ni J, Cozzi P, Hao J et al (2014) Cancer stem cells in prostate cancer chemoresistance. Curr Cancer Drug Targets 14:225–240 27. Gokalp C (2020) Cytotoxic and anti-angiogenic effects of zoledronic acid in DU-145 and PC-3 prostate cancer cell lines. Mol Biol Rep 47:7675–7683. https://doi.org/10.1007/ s11033-020-05840-6 28. Kamba T, Kamoto T, Maruo S et al (2017) A phase III mul- ticenter, randomized, controlled study of combined androgen blockade with versus without zoledronic acid in prostate cancer patients with metastatic bone disease: results of the ZAPCA trial. Int J Clin Oncol 22:166–173. https://doi.org/10.1007/ s10147-016-1037-2 29. Kattan J, Bachour M, Farhat F et al (2016) Phase II trial of weekly Zoledronate docetaxel, zoledronic acid, and celecoxib for castration- resistant prostate cancer. Invest New Drugs 34:474–480. https:// doi.org/10.1007/s10637-016-0357-4
30. Bühler H, Hoberg C, Fakhrian K, Adamietz IA (2016) Zole- dronic acid inhibits the motility of cancer stem-like cells from the human breast cancer cell line MDA-MB 231. Vivo (Brook- lyn) 30:761–768. https://doi.org/10.21873/invivo.10992
31. Wang L, Liu Y, Zhou Y et al (2019) Zoledronic acid inhibits the growth of cancer stem cell derived from cervical cancer cell by attenuating their stemness phenotype and inducing apop- tosis and cell cycle arrest through the Erk1/2 and Akt path- ways. J Exp Clin Cancer Res 38:93. https://doi.org/10.1186/ s13046-019-1109-z
32. Jensen C, Teng Y (2020) Is it time to start transitioning from 2D to 3D cell culture? Front Mol Biosci 7:33. https://doi.org/ 10.3389/fmolb.2020.00033
33. Karlsson H, Fryknäs M, Larsson R, Nygren P (2012) Loss of cancer drug activity in colon cancer HCT-116 cells during sphe- roid formation in a new 3-D spheroid cell culture system. Exp Cell Res 318:1577–1585. https://doi.org/10.1016/j.yexcr.2012. 03.026
34. Kwok TT, Twentyman PR (1985) The relationship between tumour geometry and the response of tumour cells to cytotoxic drugs — an in vitro study using EMT6 multicellular spheroids. Int J Cancer 35:675–682. https://doi.org/10.1002/ijc.29103 50517
35. Han SJ, Kwon S, Kim KS (2021) Challenges of applying mul- ticellular tumor spheroids in preclinical phase. Cancer Cell Int 21:152. https://doi.org/10.1186/s12935-021-01853-8
36. Riedl A, Schlederer M, Pudelko K et al (2017) Comparison of cancer cells in 2D vs 3D culture reveals differences in AKT– mTOR–S6K signaling and drug responses. J Cell Sci 130:203–218. https://doi.org/10.1242/jcs.188102
37. Di Zazzo E, Galasso G, Giovannelli P et al (2016) Prostate cancer stem cells: the role of androgen and estrogen receptors. Oncotarget 7:193–208. https://doi.org/10.18632/oncotarget. 6220
38. Gonnissen A, Isebaert S, Haustermans K (2013) Hedgehog signaling in prostate cancer and its therapeutic implication. Int J Mol Sci 14:13979–14007. https://doi.org/10.3390/ijms140713 979
39. Ray A, Meng E, Reed E et al (2011) Hedgehog signaling path- way regulates the growth of ovarian cancer spheroid forming cells. Int J Oncol 39:797–804. https://doi.org/10.3892/ijo.2011. 1093
40. Regan JL, Schumacher D, Staudte S et al (2017) Non-canonical hedgehog signaling is a positive regulator of the WNT pathway and is required for the survival of colon cancer stem cells. Cell Rep 21:2813–2828. https://doi.org/10.1016/j.celrep.2017.11.
41. Leão R, Domingos C, Figueiredo A et al (2017) Cancer stem cells in prostate cancer: implications for targeted therapy. Urol Int 99:125–136. https://doi.org/10.1159/000455160
42. Sari IN, Phi LTH, Jun N et al (2018) Hedgehog signaling in cancer: a prospective therapeutic target for eradicating cancer stem cells. Cells 7:208. https://doi.org/10.3390/cells7110208
43. Phi LTH, Sari IN, Yang Y-G et al (2018) Cancer stem cells (CSCs) in drug resistance and their therapeutic implications in cancer treatment. Stem Cells Int 2018:5416923. https://doi.org/ 10.1155/2018/5416923
44. Singh S, Chitkara D, Mehrazin R et al (2012) Chemoresistance in prostate cancer cells is regulated by miRNAs and Hedgehog pathway. PLoS One 7:e40021–e40021. https://doi.org/10.1371/ journal.pone.0040021
45. Huang P, Nedelcu D, Watanabe M et al (2016) Cellular cho- lesterol directly activates smoothened in hedgehog signaling. Cell 166:1176-1187.e14. https://doi.org/10.1016/j.cell.2016. 08.003
46. Riobo NA (2012) Cholesterol and its derivatives in Sonic Hedgehog signaling and cancer. Curr Opin Pharmacol 12:736– 741. https://doi.org/10.1016/j.coph.2012.07.002
47. Jeong J, McMahon AP (2002) Cholesterol modification of Hedgehog family proteins. J Clin Invest 110:591–596. https:// doi.org/10.1172/JCI16506
48. Goffinet M, Thoulouzan M, Pradines A et al (2006) Zoledronic acid treatment impairs protein geranyl-geranylation for biologi- cal effects in prostatic cells. BMC Cancer 6:60. https://doi.org/ 10.1186/1471-2407-6-60
49. Coscia M, Quaglino E, Iezzi M et al (2010) Zoledronic acid repolarizes tumour-associated macrophages and inhibits mam- mary carcinogenesis by targeting the mevalonate pathway. J Cell Mol Med 14:2803–2815. https://doi.org/10.1111/j.1582-4934. 2009.00926.x
50. Bidet M, Joubert O, Lacombe B et al (2011) The hedgehog receptor patched is involved in cholesterol transport. PLoS One 6:e23834–e23834. https://doi.org/10.1371/journal.pone.0023834
51. Lee SH, Kim R, Kang M et al (2013) Abstract 3715: Zoledronic acid inhibits cancer growth and cancer stem cell phenotypes in head and neck squamous cell carcinoma. Cancer Res 73:3715– 3715. https://doi.org/10.1158/1538-7445.AM2013-3715
52. Bizzarro V, Belvedere R, Milone MR, et al (2015) Annexin A1 is involved in the acquisition and maintenance of a stem cell- like/aggressive phenotype in prostate cancer cells with acquired resistance to zoledronic acid. Oncotarget 6:25076–25092. Doi: https://doi.org/10.18632/oncotarget.4725

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