CYR61 promotes metastasis of prostate cancer cells through the p-AKT and AR signaling pathways

doi:10.21203/rs.3.rs-5140510/v1

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Research Article

CYR61 promotes metastasis of prostate cancer cells through the p-AKT and AR signaling pathways

https://doi.org/10.21203/rs.3.rs-5140510/v1

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Background

Previous studies have shown that the cysteine-rich angiogenic inducer 61 (CYR61) contributes to prostate cancer (PCa) cell growth and survival, but its role in PCa progression is unknown. As CYR61 contains an insulin-like growth factor-binding protein domain, and insulin-like growth factor-1 (IGF1) signaling is associated with PCa progression and metastases, we evaluated their molecular interplay.

Methods

We performed siRNA knockdown of PCa cell lines including prostate carcinoma 3 (PC3) and lymph node carcinoma of the prostate (LNCaP) to investigate the role of CYR61 in PCa progression. Cells were evaluated for viability, proliferation, clonogenicity, aggregation, wound healing, and transwell migration. To examine the underlying mechanisms associated with CYR61 signaling, we assessed the activation of the PI3/AKT and MAPK pathways. Monolayer cultures treated with recombinant IGF1 at different time points were analyzed for CYR61 expression by in-cell western assays.

Results

Knockdown of CYR61 using siRNA significantly reduced cell proliferation, viability, prostasphere and colony formation, metabolic activity, and migration of PC3 and LNCaP cells, when compared with cells receiving scrambled control siRNA. In PC3 and LNCaP cells, CYR61 silencing inhibited the activation of the PI3/AKT pathway but did not impact the activity of MAPK pathways. In LNCaP cells, CYR61 silencing decreased levels of the androgen receptor (AR) splice variant AR-V7. We also found that recombinant IGF1 induced protein expression of CYR61 in a time-dependent manner in PC3 and LNCaP cells.

Conclusion

CYR61 expression was induced by recombinant IGF1, and knockdown of CYR61 decreased the capacity of metastatic PCa cells to proliferate. These results underscore the therapeutic potential of CYR61 inhibition in reducing the proliferation and metastatic capabilities of PCa cells.

CYR61

Prostate Cancer

IGF1

PI3/AKT

MAPK

Metastasis

Metabolism

Prostate cancer (PCa) is the second leading cause of cancer death in men in the United States, and the incidence rate has risen by 3% every year since 2014 (1). PCa is a heterogeneous disease; clinical risk factors include genetics, race and ethnicity, obesity, and age. As with other cancers, metastatic PCa has a poor prognostic factor (2). By 2030, it is expected that 192,500 men will be living with metastatic (PCa). Understanding the etiology and mechanisms of PCa metastasis is needed to develop chemotherapeutics to treat the disease.

The cysteine-rich angiogenic inducer 61 (CYR61, also known as CCN1) is a protein implicated in cell migration, proliferation, and apoptosis (3–8). CYR61 is a secreted extracellular matrix protein, and the specific role of this secreted form in PCa is not known. That it contains an insulin-like growth factor-binding protein domain implies that CYR61 expression or function may be impacted by insulin-like growth factor-1 (IGF1) regulation. However, this regulation in PCa has not been established. IGF1 is known to play a mitogenic role in the proliferation of PCa cells (9) and may be essential for PCa initiation (10). Circulating levels of IGF1 have been associated with resistance to various chemotherapies. IGF1 availability due to low IGF1 receptor expression is now used as a predictor of metastatic disease (11). Therefore, elucidating the interaction between CYR61 and IGF1 in PCa could provide important insights into the mechanisms driving metastasis and therapeutic resistance.

While not previously described in PCa, the relationship and potential targeting of the IGF1-CYR61 axis in metastatic osteosarcoma have been reported. Specifically, CYR61 triggers primary tumor vascularization via an IGF1 receptor (IGF1-R)-dependent epithelial to mesenchymal transition-like process (11). Because 70% of metastatic PCa patients harbor bone metastases (2), examination of IGF1-CYR61 in PCa is warranted. Here, using the metastatic PC3 and LNCaP cell lines, we investigated the role of CYR61 and its interactions with IGF1 in metastatic PCa. In addition, we revealed how the expression of the androgen receptor (AR) splice variant 7 (AR-V7), an important biomarker for metastatic disease (12), is impacted by CYR61 silencing, revealing a novel role in its function, leading to PCa cell proliferation and resistance to hormonal therapies.

Cell Lines

PCa cell lines, PC3 and LNCaP, were from the American Type Culture Collection (Manassas, VA, USA, Cat# CRL-1435, and CRL-1740, respectively) and cultured in RPMI-1640 medium (Corning, Corning, NY, USA Cat# 10-040-CM), supplemented with 10% (v/v) fetal bovine serum (FBS, Corning, Corning, Cat# MT35010CV), penicillin/streptomycin (Corning, Cat# 30-002-CI), and normocin 1G (Invivogen, San Diego, CA, USA, Cat# NC9390718). Cells were grown under 5% CO₂ at 37°C. Short tandem repeat service provided by ATCC (Cat# ATCC-135-XV) was used to authenticate the cell lines. Mycoplasma testing was conducted at least twice a year using the Lonza MycoAlertTM Mycoplasma Detection Kit (Lonza, Basel, Switzerland, Cat# LT07-218).

Antibodies

Rabbit antibodies targeting the following proteins were acquired from Cell Signaling Technology (Danvers, MA, USA): CYR61 (D4H5D) (Cat# 14479S), α/β-tubulin (Cat# 2148S), β-actin (13E5) (Cat# 4970), Phospho-AKT (Thr308) (Cat# 2965), Phospho-p44/42 MAPK (Erk1/2) (Cat# 9101S). Mouse monoclonal antibodies included CYR61 (A-10) (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-374129).

Immunoblotting

Whole-cell lysates were prepared by lysing the cell pellet in 100 µL Invitrogen™ Cell Lysis Buffer II (Thermo Fisher Scientific, Waltham, MA, USA Cat # FNN0021) on ice for 30 minutes, vortexing at 10 minutes intervals. The cell lysis buffer was supplemented with 1 mM Thermo Scientific™ PMSF Protease Inhibitor (Thermo Fisher Scientific, Cat# PI36978) and Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Cat# P178440). The cell extract was centrifuged at 13,000 rpm for 10 minutes at 4°C. The supernatant was collected, and protein concentration of the lysates was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Cat# 23225) to ensure equal loading of proteins separated on individual lanes by SDS-PAGE (NuPAGE 4–12%, Thermo Fisher Scientific, Waltham, MA, USA). Samples for immunoblot analysis were diluted in ultrapure water, Invitrogen™ 1× Bolt™ Sample Reducing Agent (Thermo Fisher Scientific, Cat# B0004), and Invitrogen™ NuPAGE™ Lithium Dodecyl Sulfate Sample Buffer (1×) (Thermo Fisher Scientific, Cat# NP0007) to a concentration of 50 µg of protein per 20 µL, then heated at 70°C for 10 minutes. Electrophoresis was followed by protein transfer to polyvinyl difluoride membranes (Millipore Sigma, Burlington MA, USA, Cat# IPFL00010). Membranes were blocked for one hour at room temperature with Intercept® Blocking Buffer (Li-Cor, Lincoln, NE, USA Cat# 927-60001) and probed overnight with appropriate primary antibodies. Membranes were then washed with TBS-Tween buffer (20 mM Tris-HCL, pH 7.6, 140 mM NaCl, and 0.2% Tween 20) three times for 5 minutes each. After washing, membranes were incubated with IRDye® 680RD Goat anti-Rabbit IgG Secondary Antibody (Licor, Cat# 926-68071) or IRDye® 680RD Goat anti-Mouse IgG Secondary Antibody (Licor, Cat# 926-68070). Washes were repeated after secondary antibody incubation. Membranes were imaged using a Li-Cor Odyssey scanner. Protein bands from at least 3 independent blots were scanned for each protein of interest, quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA, Fiji Version 1.44a), and normalized to α/β-tubulin or β-actin loading control protein bands to determine fold upregulation.

Cell viability assay

Trypan blue dye exclusion assay was employed to assess the effects of CYR61 siRNA on the viability of PC3 and LNCaP cells. Following siRNA transfection, cells (both floating and adherent) were collected, pelleted by centrifuging at 1500 rpm for 5 minutes, and resuspended in 1 mL 1× phosphate-buffered saline (PBS). After pellet resuspension, 10 µL of cell suspension was combined with 10 µL trypan blue (Thermo Fisher Scientific, Cat# MT25900CI), and live cells were counted using a Cellometer Vision CBA Image Cytometer (Nexcelom, San Diego, CA, USA). Cell viability was expressed as a percentage of the viability of the scrambled small interference RNA (siRNA) duplex (SD) control.

The CellTiter-Glo® assay was used to assess cellular proliferation and viability. Following CYR61 siRNA transfection, PC3 and LNCaP cells were seeded (5000 cells/well) in opaque-walled 96-well plates (Thermo Fisher Scientific, Cat# 12-566-620) and 100 µL complete media. Control wells containing medium without cells were used to determine background luminescence. Cells were assessed for 1 to 4 days post-transfection. After each time point, a volume of CellTiter-Glo® 2.0 Reagent equal to the volume of cell culture medium present in each well (100µl) was mixed for 2 minutes on an orbital shaker to induce cell lysis. Following this, the plate was incubated at room temperature for 10 minutes to stabilize the luminescent signal. The luminescence was recorded using an integration time of 0.25–1 second per well per manufacturer’s instructions.

RNA Interference

PC3 and LNCaP cells (50,000 cells per well) were cultured on 6-well plates and transfected after 24 hours with either 50 nM or 100 nM siRNA for up to 96 hours. The siRNA sequences used were as follows: si-CYR61 knockdown (KD, pool of three different siRNA duplexes from Santa Cruz Biotechnology, Cat# sc-39331). Cells were transfected using Interferin® siRNA transfection reagent (Polyplus-transfection®, Illkirch, France, Cat# 409-01). Scrambled siRNA duplex (SD, Dharmacon, Lafayette, CO, USA, Cat# D-001210-0105) was used as a non-targeting negative control. Protein depletion was assessed by immunoblotting.

Clonogenic Assay

PC3 and LNCaP cells were transfected with CYR61 siRNA and grown in RPMI-1640 medium supplemented with 10% FBS for 72 hours. Next, an equal number of viable transfected cells was transferred to 6-well culture plates (500 cells/well for PC3 and 1000 cells/well for LNCaP). Colony formation plates were incubated for 10 days at 5% CO₂ and 37°C. Adherent colonies were washed with 1× PBS, fixed with 3:1 (v/v) methanol:acetic acid solution for 5 minutes, washed again with 1× PBS, stained with 0.5% crystal violet for 20 minutes, and then washed gently with tap water. Images of the stained colonies were acquired using a Li-Cor Odyssey scanner and quantification was performed using the automated colony counting capability of Image J software following identical parameters for each well.

Prostasphere Formation Assay

Spheroid cultures from siRNA transfected cells were maintained using complete MammoCult™ medium (Stem Cell Technologies, Vancouver, Canada, Cat# 05620) supplemented with hydrocortisone (0.48 g/mL, Millipore Sigma-Aldrich, Cat# H0135), heparin (4 g/mL Sigma-Aldrich, St. Louis, MO, USA, Cat# H3149), and 1% penicillin/ streptomycin. PC3 and LNCaP cells were seeded at 50,000 cells per well and transfected with the various siRNAs. After 48 h, an equal number of viable cells (1000 cells/well) were harvested and resuspended 50 times in MammoCult™ medium to ensure a single-cell suspension. Cells then were seeded in 24-well untreated plates (Genesee Scientific, Morrisville, NC, Cat# 25–102) in 0.5 mL MammoCult™ medium. Prostaspheres were grown for 10 days at 37°C/5% CO₂ and visualized in a Zeiss Observer II microscope. The prostasphere area was quantified from three independent images per individual treatment using Image J software.

Wound Healing Assay

PC3 and LNCaP cells were examined for their mobility using a wound-healing assay. Following CYR61 siRNA transfection for 72 hours, cells were collected and seeded into a 24-well plate (Thermos Fisher Scientific, Cat# 09-761-146) to reach semi-confluency. Confluent transfected cells were “scratch wounded” with a P200 pipette tip. Wound closure was monitored by a Nikon Eclipse Ti microscopy. Images were taken every 12 hours over 48 hours and analyzed with ImageJ software. The average wound area relative to the initial wounding (0 hr) was determined in three independent triplicate assays and compared to control cells transfected with SD negative control.

IGF1 Induction Treatment

To determine if IGF1 increased the expression of CYR61, PC3, and LNCaP cells were induced with 100 ng/ml recombinant human IGF1 (Peprotech, Cranbury, NJ USA Cat# 100 − 11) for 30 minutes, 4 hours, and 24 hours. CYR61 protein expression levels were measured at different time points. Experimental cells were plated, and once cells reached 70% confluence, they were switched to serum-free media with 0.1% BSA for overnight starvation. Immediately after exposure to IGF1 for the designated time, the cells were collected for protein extraction or evaluated for expression.

In-Cell Western (ICW) Assay

The ICW assay was performed using the Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE, USA) according to the manufacturer's instructions. PC3 and LNCaP cells were grown in 96-well plates until they reached 60–70% confluency and then fixed with 4% paraformaldehyde at selected time intervals post-transfection or IGF1 induction for the ICW assay. Then, cells were permeabilized with 0.5% Triton X-100 for 15 minutes at room temperature and blocked with LI-COR Odyssey Blocking Solution (LI-COR Biosciences) for 30 minutes. The cells were incubated at 4°C overnight with appropriate primary antibodies. After three washes with 1× PBS, cells were stained with secondary antibodies at room temperature for 2 hours. The plates were scanned with the Odyssey CLx Infrared Imaging System (LI-COR Biosciences), and the integrated fluorescence intensities representing the protein expression levels were acquired using the software provided with the imager station (Empiria Studio 2.3, LI-COR Biosciences). The relative amount of protein was obtained by normalizing to endogenous β-actin or α/β-tubulin in all experiments.

Transwell Migration Assay

Cell migration was assessed using 24-well ThinCert™ (Greiner Bio One, Frickenhausen, Germany, Cat#662638) permeable inserts containing PET capillary pore membranes with 8.36 mm inner diameter, 10.34 mm outer diameter, 16.22 mm height, and 8 µm pore size. The assay was carried out once the transfection time ended. PC3 and LNCaP cells were trypsinized and 1 × 10⁵ live cells/ml were re-suspended in serum-free RPMI medium. While the cell suspensions were prepared, 500 µl of the chemoattractant (RPMI supplemented with 10% FBS) was dispensed into each well of the 24-well ThinCert™ plates and incubated at 37°C for 1 h. The ThinCert™ inserts were placed in the wells containing pre-warmed chemoattractant, and 1 × 10⁵ live cells/ml (200 µl from the cell suspension) were applied. Subsequently, the plates were incubated at 37°C for 48 h. The medium in the inserts was then removed and the membranes were washed twice in PBS. The PC3 and LNCaP cells that remained in the upper part of the insert were removed carefully with a PBS-soaked cotton swab. The cells that migrated or invaded towards the lower part of the chamber were fixed with 4% formaldehyde and stained with 0.5% crystal violet. The fixed cells were visualized on a Zeiss Observer II microscope and photographed. The total number of cells that migrated or invaded was counted manually using ImageJ software.

Statistical Analysis

Data are expressed as mean ± SEM from a minimum of three independent experiments. Statistical analysis was performed with GraphPad Prism version 9 (GraphPad Software, San Diego, CA, USA). Two-sample comparisons were determined using the two-tailed Student t-test. For multiple groups, we used two-way ANOVA. The p values < 0.05 were considered statistically significant.

CYR61 promotes PCa metastatic cell line viability and proliferation

We investigated the effects of CYR61 on the biological functions of metastatic PCa cell lines PC3 and LNCaP. A pool of three different CYR61 siRNA duplexes were used to transfect the cell lines. Transient silencing of CYR61 in PC3 and LNCaP cells led to robust depletion of the protein compared to cells transfected with SD control (Fig. 1A) as validated by western blot. SD cells had a relatively normal morphology with fewer floating cells and features of cell death compared to the CYR61 knockdown (KD) cell lines which showed increased cell death (Fig. 1B). Knockdown of CYR61 with 50 nM and 100 nM CYR61 siRNA oligo reduced the viability of PC3 cells by 41.7% (P = 0.0029) and 25.6% (P = 0.0171) after 72 hours, respectively, when compared to the SD control. LNCaP cells showed an even larger decrease in cell viability with a decrease of 60.03% (P < 0.0001) and 56.1% (P < 0.0001), respectively for the two doses, when compared to SD control (Fig. 1C). Because the 50 nM concentration resulted in a greater reduction in viability of PC3 and LNCaP cells, we selected this dose for subsequent experiments. To determine if there was a difference in the metabolic activity of PC3 and LNCaP cells when CYR61 is silenced, we used the CellTiter-Glo® 2.0 assay to evaluate the effect of CYR61 KD on cell proliferation by quantifying ATP, which indicates the presence of metabolically active cells. Androgen receptor (AR) is expressed in the LNCaP cell line, whereas PC3 has relatively low basal levels of AR and is unresponsive to androgen stimulation. CYR61 KD significantly inhibited the proliferation of PC3 and LNCaP cells in a time-dependent manner (Fig. 1D). These results demonstrated that CYR61 KD affected cancer growth in the highly invasive PC3 and LNCaP cell lines.

Silencing of CYR61 inhibited prostasphere formation, clonogenicity, wound healing, and migration

Next, we investigated the impact of the reduction of CYR61 on stemness properties of PC3 and LNCaP using the prostasphere assay. As depicted in Fig. 2A, PC3-SD and LNCaP-SD cells exhibited a greater number and larger size of prostaspheres compared to PC3-KD (P = 0.0023) and LNCaP-KD (P = 0.0061) cells. These findings implied that the downregulation of CYR61 diminished the ability of PC3 cells to grow as non-adherent prostaspheres.

The aggressiveness of cancer cell lines is associated with their ability to grow independently as colonies in vitro. Assessing colony formation is the standard method for measuring both proliferation and the ability of cancer cells to colonize and survive, serving as indicators of metastatic potential. When CYR61-expressing PC3 and LNCaP cells were plated, they exhibited a notably high colony count (~ 120–400) (Fig. 2B). Conversely, the knockdown of CYR61 significantly diminished the clonogenic capacity of PC3 clones and rendered LNCaP clones nearly incapable of growth or survival (P = 0.0030).

To determine whether intracellularly-suppressed CYR61 regulated PCa cell migration, we transiently reduced CYR61 (see verification of CYR61 suppression Fig. 1A) in PC3 and LNCaP cell lines and analyzed cell migration using the 2D transwell co-culture proliferation assay. Reducing CYR61 resulted in a significant decrease (P < 0.0001) in cell migration in PC3 and LNCaP cells compared to the control SD groups, as shown on stained cells on the lower surface of the membrane (Fig. 2C).

As CYR61 is known to promote metastasis in osteosarcoma and αv β3-mediated cell migration (13), we investigated whether depleting CYR61 affected the migratory ability of PC3 and LNCaP cells, as measured by the relative wound closure area at 48 hours. We found that PC3-SD cells efficiently closed the wound within 48 hours, whereas PC3-KD cells exhibited significantly poorer wound closure (P = 0.001776) (Fig. 2D). The LNCaP-SD cells exhibited a lower wound closure rate in comparison to PC3-SD cells and the LNCaP-KD exhibited significant wound closure (P = 0.000224), compared to LNCaP-SD cells.

Effect of CYR61 silencing on the AKT, MAPK, and AR signaling pathways in PC3 and LNCaP cells

We measured the effects of CYR61 silencing on the AKT, MAPK, and AR signaling pathways using western blotting. After silencing CYR61 (KD) in PC3 and LNCaP cells, levels of p-AKT in the two cell lines were significantly lower compared to the control SD group (Figs. 3A-D). These results suggest that CYR61 silencing inhibits AKT phosphorylation in PC3 and LNCaP cells.

The molecular mechanism of CYR61 silencing in AR-V7 inhibition in PCa was further investigated in AR-positive LNCaP cells (Fig. 3B). Compared with the control SD group, CYR61 KD cells showed significantly lower levels of AR-V7 (Figs. 3B, D).

IGF1 regulates CYR61 expression differently in PC3 and LNCaP cells

PC3 and LNCaP cells were induced with recombinant human IGF1 at different time points, and CYR61 expression was analyzed with the in-cell western assay (Fig. 4A-B). In PC3 cells, recombinant human IGF1 treatment increased CYR61 expression at 30-minute (P = 0.0204) and 24-hour (P = 0.0043) time points, but not at 4 hours. In LNCaP cells (Fig. 4B), IGF1 induction significantly decreased CYR61 expression at 30 minutes and 4 hours post-induction (P < 0.0001). However, CYR61 levels stabilized after 24 hours, which suggests a feedback loop increased levels of CYR61.

The connective tissue growth factor, cysteine-rich protein, and nephroblastoma overexpressed gene (CCN) family are extracellular matrix proteins with reported roles in tumor invasion (14–16). The CCN family of proteins is involved in different key molecular processes, including coordination in tumor microenvironments, tumorigenesis, and cancer metastasis (17–19). In this paper, we focused on CYR61, also known as CCN1, which has been shown to act as a tumor-promoting factor and is likely a key regulator of cancer progression (20). The IGF-binding protein domain of CYR61 is of particular interest because studies have reported that higher circulating levels of IGF1 were associated with metastatic disease and resistance to various chemotherapies (10). In this study, we focused on characterizing the effect of CYR61 and its interplay with IGF1 on metastatic PCa and potential signaling pathway mechanisms. We studied two different PCa metastatic cell lines: 1) LNCaP cells, which express AR and prostate-specific antigen (PSA), and whose biological behavior is most similar to the majority of newly diagnosed PCa cases (21) (22); and 2) PC3 cells which do not express AR or PSA, are androgen-independent (22, 23), and exhibit highly aggressive behavior, which is consistent with the biological behavior of advanced or treatment-resistant PCa. With the use of these two models, we aimed to represent a spectrum of PCa and thus represent androgen-dependent and castration-resistant tumors, respectively. Our results demonstrated that CYR61 exerted pro-survival properties on PCa cells, and its expression was further induced with IGF1.

Previously, in cellular models of breast cancer, it was found that IGF1 promotes CYR61-induced cell growth and invasion (24). In PCa, we found that IGF1 induced a significant increase in CYR61 levels in PC3 cells at 30-minute and 24-hour time points. Conversely, in LNCaP cells, IGF1 induced a significant decrease in CYR61 levels at 30-minute and 4-hour time points. Similar to what we observed in PC3 cells, Sarkissyan et al. (24), showed that in MCF7 breast cancer cells, CYR61 was upregulated significantly after 20 minutes of induction, with IGF1 demonstrating increased proliferation and invasion. A possible explanation for the decrease in CYR61 expression in LNCaP cells may be that IGF1 stimulated surface expression of the integrins α5, αV, and β1 in these cells (25). In a study investigating the effect of IGF1 stimulation on surface expression of the integrins α5, αV, and β1 in a panel of PCa cells (26), the αV subtype was reduced in LNCaP cell lines after 24 hours of IGF1 stimulation. CYR61 has been shown to bind to the cell surface through the αV integrin subtype, and αV integrin’s expression was correlated with cancer cell migration in cellular models of non-small cell lung cancer, colorectal cancer, breast cancer, and oesophageal squamous carcinoma (27). In PC3 cells, IGF1 incubation produced a considerable increase in the αV integrin subtype after both 4 hours and 24 hours, consistent with our observations for CYR61 expression. In advanced disease, individuals with high circulating levels of IGF1 have an increased risk of PCa (28). In addition, AR has been shown to directly regulate transcription of the IGF1 receptor in PCa cells (29). A second possible explanation for the decrease of CYR61 in LNCaP cells is aberrant AR activation. Induction of these cells with recombinant human IGF1 and an increase in CYR61 expression can implicate a regulatory mechanism underlying AR expression through IGF1-mediated signaling pathways in PCa progression. Our findings suggest a potential novel interaction between AR and IGF1 signaling pathways that can contribute to PCa. Together, our observations indicate for the first time that CYR61 expression can be induced by IGF1 in the context of PCa, showing that this protein is highly sensitive to growth factor induction. The upregulation of CYR61, which mediates cell proliferation, migration, and invasion, may result in increased metastasis. This result is relevant to patients with high IGF1 circulatory levels who can present higher PCa risk and reduced survival.

A novel aspect of our study was the investigation of the ability of CYR61 to induce stemness. For that purpose, we optimized a prostasphere assay on CYR61 (KD) knockdown cells. This assay serves as the standard method for assessing the self-renewal capacity of cancer-stem like cells (CSC) in cell cultures (30). In addition, this assay relies on the premise that only CSCs can thrive and proliferate without anchorage in the absence of serum. Aggressive cell lines, such as PC3, in which expression of mesenchymal/stem cell molecular markers are exacerbated, upregulate levels of CYR61 (Supplementary Fig. 1). In cellular models of breast cancer, it has been suggested that mesenchymal-transformed non-invasive cells, such as MCF-7, show increased invasiveness and elevated CYR61 expression (31). In our prostaspheres models, we show for the first time that CYR61 knockdown impaired the formation of these spheres in PC3 and LNCaP cells. Similarly, in models of pancreatic carcinogenesis, CYR61 silencing reversed the epithelial-to-mesenchymal transition, blocked the expression of stem-cell-like traits, and inhibited migration (32).

We investigated whether the CYR61 upregulation due to IGF1 induction was mediated through the PI3K/AKT pathway, which is one of the central pathways in IGF1-induced PCa growth (33). CYR61-KD PC3 and LNCaP cells showed a significant decrease in p-AKT levels, but not AKT when compared to CYR61-SD control cells. The MAPK pathway, which is the other key downstream pathway from IGF1 was also assessed (34). CYR61-KD PC3 and LNCaP cells did not show significant changes in levels of p-MAPK or MAPK. Together, these data suggest that IGF1-mediated CYR61 expression is intrinsically mediated through the PI3K/AKT signaling pathway and not the MAPK signaling pathway.

The AR signaling pathway regulates PCa cell proliferation and apoptosis (35). An important mechanism through which PCa adapts to treatments targeting AR signaling is the constitutively active AR splice variants, especially AR-V7. The AR-V7 splice variant has been studied extensively, but its role in regulating the metastatic progression of castration-resistant PCa (CRPC) remains unclear (36). Our results showed that, in LNCaP cells, CYR61 knockdown significantly decreased AR-V7 levels. These results demonstrate that CYR61 might be a direct target or downstream effector of AR-V7 and may be a new therapeutic target for advanced PCa. In addition, our data provide molecular insights into the role of AR-V7 in driving the PCa metastatic progression mediated by CYR61.

Our findings implicating IGF1 induction in CYR61 upregulation have potential translational applications (37). Pooled analyses examining the associations of anthropometric, behavioral, and sociodemographic factors with circulating concentrations of IGF1 in 16,024 men from 22 studies reported low expression of the IGF1-R in Hispanic/Latino (H/L) men with PCa. Among H/L men, high serum levels of IGF1 were associated with the development of PCa. In addition, low IGF1-R expression predicted metastatic disease (10). Among African-American men, variants in IGF1 were associated with PCa risk (38). Furthermore, circulating levels of IGF1 are hereditary and associated with resistance to various chemotherapies, and their levels are modulated by Vitamin D (39, 40). Consequently, increased IGF1-induced CYR61 activity through the classical PI3K/AKT signaling pathway may be a mechanism by which tumors progress to a more aggressive and invasive phenotype. Studies are needed to confirm these findings and to determine if targeting CYR61 may be used as an alternative therapeutic option for advanced PCa.

An important limitation in our study should be acknowledged. While our study focused on the role of CYR61 and its interplay with IGF1 in two metastatic PCa cell lines, the findings may not fully capture the complexity of PCa progression in vivo. Although representative of androgen-dependent and castration-resistant tumors, the use of LNCaP and PC3 cell lines may not reflect the full heterogeneity of PCa seen in patients. The cell lines have inherent limitations, such as differences in genetic background and cellular behavior that may not accurately mimic the tumor microenvironment or the molecular interactions within a living organism. Therefore, while the findings provide valuable insights, further research is needed to explore these additional pathways and validate the results in more complex and clinically relevant models.

We showed that CYR61 played a role in the survival, migration, proliferation, cell metabolism, and self-renewal capacity of stem-like cell populations in the PC3 and LNCaP cell lines. Furthermore, CYR61 expression was induced by IGF1 differentially in these cell models. IGF1-mediated CYR61 expression was mediated through the PI3K/AKT signaling pathway in PC3 and LNCaP cells and associated with the levels of AR-V7 in our cellular models (Fig. 5). Our results suggest a potential mechanism that drives PCa metastatic disease, and targeting CYR61 in metastatic disease could play a role in PCa treatment.

AR	Androgen Receptor
CCN	Connective tissue growth factor, Cysteine rich protein and Nephroblastoma overexpressed gene
CSC	Cancer Stem-Like Cell
CYR61	Cysteine-Rich Angiogenic Inducer 61
H/L	Hispanic/Latino
ICW	In-Cell Western
IGF1	Insulin-Like Growth Factor-I
IGF1-R	Insulin-Like Growth Factor-Receptor
KD	Knockdown
LNCaP	Lymph Node Carcinoma of the Prostate
MAPK	Mitogen-Activated Protein Kinases
p-AKT	Phosphorylated Protein Kinase B
p-MAPK	Phosphorylated Mitogen-Activated Protein Kinases
PBS	Phosphate-Buffered Saline
PC3	Prostate Carcinoma 3
PCa	Prostate Cancer
PFA	Paraformaldehyde
PI3/AKT	Phosphatidylinositol 3-Kinase/Protein kinase B
PSA	Prostate Specific Antigen
SD	Scramble siRNA Duplex
siRNA	Small Interference RNA
TBS	Tris Buffer Saline

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

The datasets analyzed and the materials used during the current study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

G.L.O.H. research was supported by the T32 CA221709/CA/NCI NIH and the Burroughs Wellcome Fund-Postdoctoral Enrichment Program. S.H. was supported by an American Cancer Society-Fred Ross Desert Spirit Postdoctoral Fellowship (PF‐21‐184‐01‐CSM).

Authors' contributions

G.L.O.H. designed and performed the experiments, analyzed data, and wrote the original draft. C.P. performed experiments, S.H. assisted in data acquisition and analysis. S.N. and M.L. supervised all experiments and supported the study. Y.R.L assisted in the design and provided cell lines for the study. All authors revised and approved the submitted version of the manuscript.

Acknowledgments

G.L.O.H would like to thank Dr. Rick Kittles and the members of Dr. Susan L. Neuhausen, Dr. Mark LaBarge, and Dr. Kittles laboratories for their helpful discussions and technical assistance during the conception of this project. G.L.O.H would also like to thank the support received from the Postdoctoral Training Office, T32 Cancer Metabolism Training Program, the T32 committee, and the Department of Population Sciences at the City of Hope. Research reported in this publication included work performed in Light Microscopy and Digital Imaging Shared Resource supported by the National Cancer Institute of the National Institutes of Health under grant number P30CA033572. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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No competing interests reported.

SupplementalFigure1.pdf
Supplemental Figure 1. Protein expression levels of CYR61 were significantly increased in metastatic PCa cells.
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CYR61 promotes metastasis of prostate cancer cells through the p-AKT and AR signaling pathways

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

BACKGROUND

METHODS

Cell Lines

Antibodies

Immunoblotting

Cell viability assay

RNA Interference

Clonogenic Assay

Prostasphere Formation Assay

Wound Healing Assay

IGF1 Induction Treatment

In-Cell Western (ICW) Assay

Transwell Migration Assay

Statistical Analysis

RESULTS

CYR61 promotes PCa metastatic cell line viability and proliferation

Silencing of CYR61 inhibited prostasphere formation, clonogenicity, wound healing, and migration

IGF1 regulates CYR61 expression differently in PC3 and LNCaP cells

DISCUSSION

CONCLUSIONS

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1