Abstract
Androgen deprivation therapies aimed to target prostate cancer (PrCa) are only partially successful given the occurrence of neuroendocrine PrCa (NEPrCa), a highly aggressive and highly metastatic form of PrCa, for which there is no effective therapeutic approach. Our group has demonstrated that while absent in prostate adenocarcinoma, the αVβ3 integrin expression is increased during PrCa progression toward NEPrCa. Here, we show a novel pathway activated by αVβ3 that promotes NE differentiation (NED). This novel pathway requires the expression of a GPI-linked surface molecule, NgR2, also known as Nogo-66 receptor homolog 1. We show here that NgR2 is upregulated by αVβ3, to which it associates; we also show that it promotes NED and anchorage-independent growth, as well as a motile phenotype of PrCa cells. Given our observations that high levels of αVβ3 and, as shown here, of NgR2 are detected in human and mouse NEPrCa, our findings appear to be highly relevant to this aggressive and metastatic subtype of PrCa. This study is novel because NgR2 role has only minimally been investigated in cancer and has instead predominantly been analyzed in neurons. These data thus pave new avenues toward a comprehensive mechanistic understanding of integrin-directed signaling during PrCa progression toward a NE phenotype.
Introduction
Integrins are transmembrane receptors composed of two non-covalently linked subunits (α and β) and, as indicated by various studies, show altered distribution during prostate cancer (PrCa) progression1,2,3. The αVβ3 integrin is usually present at low levels in normal human prostate cells but is highly expressed in advanced PrCa; it promotes invasion and adhesion of cancer cells to extracellular matrix proteins, such as vitronectin3,4. The activation state of the αVβ3 integrin, the affinity/avidity for cognate ligands, is regulated by Kindlin-2 (K2, Mig-2, FERMT2)5,6,7, which interacts with the cytoplasmic tail of the β3 subunit8,9,10. K2 is expressed in many different cell types and plays an essential role during integrin-dependent interaction between cells and the extracellular matrix11.
Recently, we have reported that the αVβ3 integrin is highly expressed in human and mouse neuroendocrine prostate cancer (NEPrCa) but absent in prostate adenocarcinoma (ADPrCa)12,13. In contrast, another αV integrin, αVβ6, is present in ADPrCa14 but is negligible in NEPrCa13. NEPrCa is a highly aggressive and metastatic subtype of PrCa that typically develops from subsets of castrate-resistant PrCa (CRPrCa) cells15. NEPrCa develops either de novo or through the acquisition of alterations in pre-existing epithelial tumors in response to androgen deprivation therapies15,16,17. The NE phenotype appears to result from cells that do not express androgen receptor (AR) or prostate-specific antigen (PSA) but instead express neuron-specific proteins, such as synaptophysin (SYP), neuron-specific enolase (NSE), and chromogranin A (CHGA)18,19. These aberrations activate pro-tumorigenic pathways independently from those downstream of the AR20,21. A recent study suggests that other changes, in addition to AR signaling loss, are necessary for neuroendocrine differentiation (NED) to take place22. The role of the αVβ3 integrin in NEPrCa has not been investigated.
The Nogo receptor family is formed by three structurally related molecules: NgR1, NgR2, and NgR3. NgR2 core protein (45 kDa), in this study its sialylated form (48 kDa), or glycosylated forms (65 kDa) are predominantly detected23. NgRs are glycosylphosphatidylinositol (GPI)-anchored receptors that lack both the transmembrane and the intracellular domains24,25. This protein family is characterized by eight leucine-rich repeats (LRR) flanked by the N- and C-terminal cysteine-rich regions. The C-terminal LRR domain is connected to the GPI-anchor for the membrane attachment via a stalk region24,25. Despite its name, NgR2 does not bind to Nogo26, but it is known to bind to myelin-associated glycoprotein (MAG)23,25. Members of the NgR family form a signal transduction complex with the nerve growth factor receptor p7524,27 and LINGO-128 that activates RhoA29. RhoA is a member of the Ras superfamily of small GTPases that, in cancer, regulates cytoskeletal dynamics to mediate cell migration30. In PrCa, elevated RhoA levels have been associated with aggressive disease and decreased disease-free survival after radical prostatectomy31. In addition, enzalutamide-resistant PrCa cells express higher levels of RhoA compared to their enzalutamide-sensitive counterparts32. Finally, activation of RhoA by the neuropeptide bombesin stimulates PC3 cell migration33.
Here we demonstrate that the αVβ3 integrin, known to be upregulated in NEPrCa13, increases the levels of a GPI-anchored receptor called NgR2 (Nogo-66 receptor homolog 1) in NEPrCa cells. The role of the NgR protein family, to the best of our knowledge, has been minimally investigated in cancer34,35,36, as it has been predominantly studied in neurons. Specifically, NgR2 is reported in a correlative study to be associated with Hodgkin lymphoma34. We show here that NgR2 is significantly upregulated in NEPrCa patients’ tumors and NE cell lines and is co-expressed with NE markers in NEPrCa patient-derived xenografts (PDXs), and different NE mouse models. Moreover, from a mechanistic point of view, we show that NgR2 promotes NE differentiation, anchorage-independent growth, and cell motility. We also show that the αVβ3 integrin immunoprecipitates with NgR2. Finally, the αVβ3 integrin has to be activated by K2 in order to induce NgR2 upregulation. Our results show that NgR2 is a novel effector of the αVβ3 integrin that promotes NED in PrCa and contributes to the highly motile phenotype of NEPrCa.
Materials and methods
Cell lines
Culture conditions for the PrCa cell lines (C4-2B, LNCaP, PC3) have been previously described4,37,38. NCI-H660 cells were grown following ATCC instructions. PC3 cells were transfected using lentivirus shRNA clones to: Kindlin-2, clone TRCN0000128058 (which targets a coding sequence of K2); and non-targeting scrambled control SHC002 (purchased from Sigma). The lentivirus-mediated shRNA gene knockdown procedures were previously described in39,40. LNCaP cells were used for CRISPR/Cas9-mediated knockout of Kindlin-2 (FERMT2) as previously described41. Culture conditions for the PrCa cell lines 22Rv1 and VCaP were previously described42.
Generation of Kindlin-2 Knockout cell lines using electroporation
The sgRNA pool of 3 guide RNAs (G*A*C*GGGAUAAGGAUGCCAGA, C*G*C*GGUUCAGGUCCGUCACA and A*G*G*CGUGAUGCUUAAGCUGG with their respective Synthego modified EZ scaffolds) targeting FERMT2 and the scramble control were obtained from Synthego. The sgRNA pellets were rehydrated in 1X TE buffer (provided by Synthego) to make a stock of 100 μM. A working solution of 30 μM sgRNA was made (in nuclease free water) fresh before electroporating the cells. For every reaction, the Ribonucleoprotein (RNP) complex was assembled by adding 3 μl of30 μM sgRNA to 0.5 μl of 20 μM Cas9 (provided by Synthego) at a ratio of 9:1 in 3.5 μl resuspension buffer R (Neon Transfection System; Invitrogen Basel, Switzerland) and incubated for 10 min at room temperature.
Electroporation of LNCaP cells was achieved by an implemented electroporation device system according to manufacturer’s instructions (Neon Transfection System; Invitrogen, Basel, Switzerland). The Neon Transfection System 10 μL kit was used for the transfection of human prostate cancer cells. Cells were cultured 48 h before electroporation and harvested at nearly 80% confluency. Cells at a density of 2 × 105 were washed with PBS and resuspended in 5 μl resuspension buffer R (Neon Transfection System; Invitrogen Basel, Switzerland). Within 15 min of resuspension, the cells were added to the tube containing RNP and the cell-RNP complex was electroporated with the Neon Transfection System. Per electroporation, 2 × 105 cells were taken up in a 10 μl Neon tip using the Neon Transfection System pipette (Invitrogen). The electroporation was performed by applying 3 pulses at 1450 Volts for 10 ms to PC3 cells and 2 pulses at 1200 Volts for 20 ms to LNCaP cells. Control cells were incubated with the resuspension buffer without the sgRNA and electroporated at the same settings. After electroporation, the cells were seeded in a 6-well plate by adding 2.5 ml DMEM (Cytiva) with 10% FBS without antibiotic supplements. The cells were cultured for 48–72 h and subsequently proceeded for further analysis. Western blot analysis was used to assess the Kindlin-2 KO efficiency in all cell lines.
Antibodies
The following antibodies (Abs) were used: for the immunoblotting (IB) analysis, rabbit monoclonal Abs against the αVβ3 integrin (13166S, Cell Signaling) and Aurora Kinase A (14475S, Cell Signaling), polyclonal goat Abs against the αVβ6 integrin (AF2389, R&D system) and NgR2 (AF2776, R&D system), rabbit polyclonal Abs against calnexin (CANX, sc11397, Santa Cruz), actin (a2066, Sigma), RhoA (sc-179, Santa Cruz), TSG101 (Abcam, ab30871), mouse monoclonal Abs against RhoA (sc-418, Santa Cruz), NSE (LS-C197136, LSBio), Kindlin-2 (MAB2617, Millipore), were also used. For immunohistochemical analysis, rabbit monoclonal Ab against the β3 integrin (13166S, Cell Signaling), rabbit polyclonal Abs against SYP (PA1-1043, Invitrogen), and NgR2 (PA5-98577, Invitrogen) were also used. Rabbit IgG (I5006, Sigma) was used as negative control. For immunoprecipitation, rabbit polyclonal Ab against NgR2 (PA5-98577, Invitrogen), rabbit monoclonal Ab against the β3 integrin (13166S, Cell Signaling), mouse monoclonal Abs against the β6 (62A1) and β1 integrins (NBP2-52708, Novus) were used. For the adhesion assay, the αVβ3 integrin (LM609, Millipore MAB1976), and the non-immune mouse IgG (02-6502, Thermo Fisher) were used.
Immunoprecipitation
PC3 cells were lysed with lysis buffer (50 mM Tris–HCl pH 7.2, 150 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, 1 mM Na4O7P2, 50 mM NaF, 0.01% aprotinin, 4 μg/ml pepstatin, 10 μg/ml leupeptin, 1 mM PMSF, 1 mM CaCl2, 1 mM MgCl2, 1 μM Calpain inhibitor) and pre-clearing was performed by two consecutive incubations with protein G-Sepharose (17061801, Cytivia) at 4 °C for 30 min. Binding to specific Abs was performed by incubation at 4 °C overnight, followed by incubation with protein G-Sepharose at 4 °C for 3 h. After six washes with lysis buffer, immunocomplexes were resuspended in 1X reducing Laemmli buffer and separated by SDS-PAGE. All western blotting films were developed using the Protec Optimax developer system. The film’s images were acquired using a Microtek ArtixScan M2 and processed using the Microtek Scan Wizard Pro V8.20 software….
