3.1 CIgG is frequently expressed in PCa and CIgG transcripts with unique patterns of VHDJH rearrangements are found in prostate cancer cells.
CIgG mRNA levels were significantly higher in PCa samples than in adjacent normal samples (Fig. 1A). We performed IHC to investigate CIgG protein expression in prostate tissues. IHC analysis indicated that CIgG staining in tumor tissues was mostly cytoplasmic in basal cells, Furthermore, we explored the staining profile of CIgG in normal prostate tissue. All 5 cases of benign prostate hyperplasia exhibited weak or negative CIgG staining when compared with that in the prostate cancer tissues (Fig. 1B), Moreover, among all the specimens, CIgG staining was stronger in specimens with either high Gleason score (p = 0.044) or advanced clinical stage(p = 0.027) (Fig. 1C-F).
To determine whether IgG was produced by the cancer cells themselves or was obtained by extracellular uptake, we determined the transcription of IgG heavy chain in LNcap, PC3 and DU145 cells by RT-PCR using primers for both constant and variable regions. The results showed that the transcript of IgG heavy chain was significantly expressed in the three cancer cell lines (Fig. 1G, Figure S1, Table S4). Subsequently, the sequence features of these VHDJH rearrangements were analyzed via comparison with the best matching functional germline IgVH, IgDH and IgJH genes. The results clearly revealed that, like the B-Igs, all CIgG transcripts displayed classical and functional VHDJH rearrangement patterns. However, unlike B cell-derived IgVH, which has great diversity, several sets of VHDJH rearrangements were frequently present in the cell lines and even shared among different the cell lines, IGHV4-30/IGHD4-11/IGHJ4 were observed in 8/8 in PC3 cell samples; IGHV3-7/IGHD3-10/IGHJ5 were observed in 5/8 in DU145 samples; IGHV3-15/IGHD3-3/IGHJ4 were observed in 4/8 in LNcap samples. The prostate cancer-VHDJH rearrangements showed restricted VH, DH, and JH usage and unique VHDJH patterns, such as VH3 which was frequently present (16/24 VHDJH rearrangements analyzed in this study), especially VH3-7, which was expressed in DU145 (5/8), and LNcap (2/8) cells; moreover, among germline IGHJ1-6 genes, only IGHJ4 (16/24) and IGHJ5 (8/24) were frequently expressed, however, IgDH showed diversity in each cell line, DH4-11 rearrangement was observed in PC3 cells; DH3-10, DH2-15 and DH1-26 were observed in DU145 cells; and DH3-3, DH2-21 and DH5-12 were observed in LNcap cells. (Fig. 1G, Figure S1, Table S4). In addition, to enhancing IgG affinity, B cell-derived IgVH of IgG was usually hypermutated. Thus, we analyzed the mutation pattern in prostate cancer-derived IgVH, and compared the sequence homology among VHDJH rearrangements from three cancer cell lines. We found that prostate cancer-derived IgVH only showed a low frequency of mutation (Fig. 1G, Figure S1, Table S4). Furthermore, the same mutated points were frequently shown among different VHDJH rearrangements, resulting in high homology between VHDJH rearrangements in IgVH (Fig. 1G, Figure S1, Table S4). The results suggested that the conserved domain of IgVH as well as that of IgHJ4 and IgHJ5 may support the common functions of different VHDJH rearrangements in prostate cancer cells, but IgDH determines the unique biological activity of each VHDJH rearrangement.
3.2 CIgG is essential for the anchorage of cancer cells to the extracellular matrixand and for cell-cell adhesion, and knockdown of IgG reduces the proliferation, migration and invasion of prostate cancer cells
To investigate the functional relevance of CIgG-mediated induction of the malignant phenotype in prostate cancer cell lines, we performed cell migration and invasion assays. We found that CIgG knockdown resulted in an obvious reduction in the migratory, invasive and proliferative ability of cells (Fig. 2A-D). Conversely, prostate cancer cells with ectopic CIgG expression had increased cell migration and invasion (Fig. 2E and 2F).
Next, we performed animal studies to investigate the influence of CIgG on tumor growth in vivo. The in vitro results were supported, BALB/c nude mice that were administered subcutaneous injections of PC3 and DU145 cells with CIgG knockdown had significantly reduced tumor volme and weights compared with those in mice injected with prostate cancer cells carrying the empty vector (Fig. 2G-J).
3.3. Cigg Cells Displayed More Csc-like Characteristics
To further analyze whether CIgGhigh cells have CSClike characteristics, we performed colony formation, sphere formation and drug-resistance assays to determine their proliferation, selfrenewal and drug-resistance abilities in vitro. PC3 cells with CIgGhigh expression displayed significantly higher colony forming and sphere forming efficiency and greater resistance to paclitaxel than CIgG−/low cells (Fig. 3A-C). To confirm that CIgGhigh cells have tumor initiating abilities, CIgGhigh and CIgG−/low cells purified from PC3 cells were used to perform tumorigenicity assays in NOD/SCID mice. As few as 500 purified CIgGhigh cells showed higher tumor formation ability than CIgG−/low cells (Fig. 3D). Tumors were observed in 50% (3/6) of the CIgG−/low mice compared to 83.3% (5/6) of the CIgGhigh mice. Moreover, tumor volume and weight in the CIgG−/low group were lower than those in the CIgGhigh group (Fig. 3D-E).
3.4. Cigg Is An Ar-repressed, Adt-inducible Gene
Interestingly, we analyzed another set of samples that consist of tissue specimens from 5 patients with prostate cancer before and after receiving ADT, collected from Peking University People`s Hospital (Beijing, China). CIgG was increased in prostate tumors from patients who had undergone ADT compared with the corresponding levels in the same patients before ADT treatment (Fig. 4A-B). Moreover, CIgG was significantly elevated in the prostate PDX model subjected to castration in the GEO prostate cancer datasets (Fig. 4C). We also found that cells treated with the AR ligand DHT had lower levels of CIgG, and this effect occurred in a dose-dependent manner (Fig. 4D).
Cytoplasmic CIgG was increased in prostate tumors from patients who had received ADT compared to those from patients before ADT treatment. In addition, we found that AR-negative PC3 and DU145 cells, which readily metastasize to bone[19, 20], had higher CIgG expression levels than cell lines that do not metastasize, such as AR-positive LNCaP, and C4-2 (Fig. 4E)
To assess whether the abundance of CIgG was mediated by ADT, we validated the expression of CIgG in AR-positive LNCaP and C4-2 cells relative to the AR signaling response. In AR+ LNCaP and C4-2 cells, transient ablation of androgen led to increases in both mRNA and protein expression of CIgG (Fig. 4F-H); moreover, LNcap cells treated with the AR ligand DHT had lower levels of CIgG, and the downregulated expression could be rescued by the addition of the AR antagonist ARN509 (Fig. 4I); in addition, C4-2 cells treated with ARN509 showed upregulated CIgG expression (Fig. 4J). All these data indicate that AR itself is a repressor of CIgG expression.
To further confirm the biological function of AR on CIgG, we performed a luciferase reporter assay. The relative luciferase signals from the reporter plasmid into which the CIgG gene promoter had been inserted were significantly reduced by cotransfection with the AR plasmid (Fig. 4K).
3.5 CIgG associates with SOX2 expression and contributes to CRPC NE progression
It has been suggested that AR directly represses SOX2 in castration-resistant prostate cancer cell lines , The mean expression correction was validated in TCGA prostate cancer datasets, showing that SOX2 mRNA expression correlates inversely with AR (Fig. 5A-B). Our AR chromatin-IP first showed that AR binds the SOX2 promoter in castration-sensitive LNcap cell line (Fig. 5C). In addition, we found that AR knockdown was able to increase the expression of SOX2 (Fig. 5E). SOX2 is a critical TF that has been implicated in resistance to antiandrogen therapy [22, 23]. We hypothesized that CIgG stimulates malignant progression through the upregulation of SOX2 after ADT. CIgG expression was positively associated with SOX2 expression, as confirmed with TCGA prostate cancer datasets (Fig. 5D). We next examined whether CIgG abundance is upregulated by SOX2 in prostate cancer cell lines. Using SOX2-specific siRNA in C4-2 and DU145 cells, we observed a reduction in CIgG (Fig. 5F).
Taken together, the data demonstrate that CIgG can be induced by ADT though SOX2.
Here, we observed that knockdown of CIgG in C4-2, and DU145 cells consistently led to a decrease in the phosphorylation levels of MAPK/ERK and AKT (Fig. 5H). The data suggest that CIgG promotes MAPK/ERK and AKT activation. MEK/ERK and AKT signaling pathways play an important role in treatment resistance to facilitate PCa progression to CRPC[14, 24]. Moreover, MAPK/ERK and AKT are well-known drivers of CRPC[13, 15], and NSE, an NEPC (neuroendocrine prostate cancer) marker, levels decrease as well (Fig. 5H). These findings suggest CIgG-mediated MAPK/ERK and AKT activation as a mechanism of resistance to antiandrogen therapy.
Western blot analysis corroborated that the expression of E-cadherin (CDH1), a protein negative-related to tumor invasion, was clearly increased, and the expression of proteins positiverelated to tumor invasion, such as N-cadherin, vimentin and snail, was reduced (Fig. 5G) by CIgG knockdown.
We also examined the effect of RP215 on established xenograft models Following injection of RP215 at 5 mg/kg around the tumor, we observed significant inhibition of the growth of the treated tumors compared with that of control tumors treated with mIgG. At the termination of the experiment, the size and weight of tumors in the RP215 treated group were significantly lower than those in control group, and IHC analyses showed that RP215 inhibitor-treated tumors had reduced CIgG, pERK, pAKT and vimentin expression (Fig. 5I-L).