Cancer-driven IgG promotes the development of castration-resistant Prostate Cancer though the SOX2-CIgG pathway

Although Androgen deprivation therapy (ADT) is the initial treatment strategy for prostate cancer, recurrent castration-resistant prostate cancer (CRPC) eventually ensues. In this study, cancer-derived immunoglobulin G(CIgG) was found to be induced after ADT, identifying CIgG as a potential CRPC driver gene. Methods: The expression of CIgG and its clinical signicance in prostate cancer tissue was analyzed by TCGA database and immunohistochemistry. Subsequently, the sequence features of prostate cell line (LNcap, DU145, PC3) VHDJH rearrangements were analyzed via comparison with the best matching functional germline IgVH, IgDH and IgJH genes. We also assessed the effect of CIgG on the migratory, invasive and proliferative abilities of prostate cancer cells in vitro and vivo. Cells with high CIgG expression (CIgG high ) and low CIgG expression (CIgG -/low ) from the PC3 cell line were sorted by FACS using a CIgG monoclonal antibody named RP215, then, suspended microsphere, colony formation and drug-resistant assays were performed. A NOD/SCID mouse tumor xenograft model was developed for the study of the tumorigenic effects of the different cell populations. The AR-SOX2-CIgG signaling pathway was validated by immunohistochemistry, immunouorescence, qRT-PCR, Western blot, luciferase and ChIP assays and bioinformatics analyses. Finally, we investigated the effect of RP215 inhibition on the progression of prostate cancer in vivo using a Babl/c nude mouse xenograft model. Results:


Background
Prostate cancer (PCa) is largely dependent on androgens, which fuels tumor survival, and ADT has become the standard treatment for locally advanced or metastatic PCa. However, despite initial responses, most PCa cases eventually recur after rst-line ADT as CRPC. Although the current therapies for CRPC, for example, chemotherapy based on docetaxel or next-generation androgen receptor pathway inhibitors (ARPIs) such as enzalutamide (ENZ) and abiraterone (ABI), extend survival, durable complete responses are rare and these therapies also eventually fail [1,2]. Therefore, identifying unrecognized molecular mechanisms that are induced by ADT and drive the development of CRPC is critical, and could lead to more curative therapies for CRPC. Immunoglobulins (Igs) are a family of immune molecules that play essential roles in immunne responses. In recent decades, our laboratory and other research groups have revealed that cancer cells can also express Ig, and this speci c type of Ig is called cancer-derived immunoglobulin G (CIgG) [3][4][5][6][7][8][9].
Fortunately, A monoclonal antibody called RP215 recognizes glycosylated epitope of the CIgG heavy chain with little cross-reactivity to B-cell-derived Ig [10], therefore providing an unparalleled advantage over regular anti-human IgG antibodies in studying CIgG. Related studies revealed that CIgG contributes to the malignant behaviors of cancer cells by displaying stem cell-like features [3,11,12] and prompting EMT which casuses metastasis. Interestingly, our studies also demonstrated that CIgG can be induced by ADT, and we hypothesized that CIgG can participate in the oncogenic network which is suppressed by AR signaling.
An earlier study demonstrated that ADT increases the expression of SOX2. Herein, we assume that CIgG is a novel factor regulated by the AR-SOX2 signaling pathway. CIgG is associated with the malignant behavior of prostate cancer, and its overexpression induced by ADT can promote the development of CRPC by increasing the activity of MARK/ERK and AKT which are well-established drivers of CRPC [13][14][15][16]. In addition, several lines of evidence have connected the epithelial-to-mesenchymal transition (EMT) to CIgG, Finally, using a Babl/c nude mouse xenograft model, we demonstrate that RP215 inhibits the tumor progression of PCa in vivo.
Collectively, our studies reveal a novel AR-SOX2-CIgG pathway that promotes the development of CRPC.
We propose that CIgG is an attractive biomarker and therapeutic target for PCa.

Tissue microarrays, samples and immunohistochemistry analysis
A tissue microarray (TMA) was obtained from Shanghai Outdo Biotech (Shanghai, China), this TMA consisted of 72 primary tumors (Gleason score was available in 66 cases) and 5 normal prostate tissues.
In addition, 19 primary tumor samples, were obtained from the BioBank of Peking University People's Hospital and used for immunohistochemical analysis. In summary, 91 primary tumor cases, and 5 normal tissue cases, with valid information were used for the IHC analysis in Fig. 1B-D. Another 5 patientmatched pre-ADT biopsies and 5 post-ADT prostatectomy specimens were detected by immunohistochemistry (Fig. 4A). The procedures for IHC staining and calculation of CIgG staining scores have been described previously [17], Low CIgG expression was de ned as a score of 0-3, and high expression was de ned as a score of 4-9.

Cell culture and reagents
The prostate cancer cell lines LNcap, C4-2, PC3, DU145, and 293T were obtained from ATCC and maintained by Peking University People`s Hospital. 293T cells and prostate cancer cell lines were cultured in Dulbecco's modi ed Eagle medium (DMEM) (HyClone, Logan, UT, USA) and minimum essential medium (HyClone), RPMI 1640 (Gibco) respectively in a humidi ed atmosphere of 5% CO2 at 37 °C, supplemented with 10% FBS (HyClone) and 1% penicillin-streptomycin (HyClone). All cell lines used in this study were regularly authenticated by morphologic observation and con rmed to be free of mycoplasma contamination.

Cell migration and invasion assays
Cell migration and invasion were evaluated using Transwell invasion assays with or without Matrigel. To assess the effect of CIgG on cell migration and invasion, 1 × 10 5 cells were plated into the upper chamber of a 24-well Transwell or Matrigel chamber with 8-µm pores (Corning, NY, USA).
For cell migration assays, PC3 and DU145 cells were incubated for 18 h prior to the assay. For cell invasion assays, PC3 and DU145 cells were incubated for 24 h. The nonmigrating cells were removed from the upper surface of the membrane. The cells on the lower surface of the wells were stained with 1% crystal violet and counted in 6 randomly selected microscopic elds (200 × magni cation).

CCK-8 assays
The effect of CIgG on cell proliferation was evaluated using the Cell Counting Kit-8(CCK-8) assay (Dojindo, Kumamoto, Japan). Brie y, 2,000 cells in 150 µl of medium were seeded onto 96-well plates. The absorbance of each well at 450 nm was measured at ve different time points. Prior to all absorbance measurements, the medium in each well was replaced with 100 µl of complete medium supplemented with 10% CCK-8 solution, and the cells were incubated for 2 h.

Colony formation assays
Cells were plated in 6-well plates at a density of 500 cells per well and cultured for 2 weeks. Then, the colonies were xed with 4% paraformaldehyde for 20 min, stained with a 0.5% crystal violet solution for 20 min, and counted.

Xenograft tumor model
The animal studies were approved by the Institutional Committee of Peking University People's Hospital. Male NOD/SCID and BALB/c nude mice that were 4-6 weeks old were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The mice were allowed to acclimate for 1 week after arrival. For the tumorigenicity assays, male BALB/c nude mice were subcutaneously injected with 1 × 10 6 CIgG shRNA vector-transfected PC3 cells in 50% Matrigel (BD Biosciences).
The cultured PC3 cells were stained using RP215, and CIgG High or CIgG −/low cells were sorted by FACS.
Five hundred cells CIgG High or CIgG −/low cells were transplanted subcutaneously into the mammary fat pads of NOD/SCID mice (Vital River, Beijing, China) in 50% Matrigel (BD Biosciences). After 5 weeks, the NOD/SCID mice were sacri ced and the number of tumors formed was measured.
For RP215 treatment, BALB/c nude mice were injected with PC3 cells for 20 days, and then the mice were sacri ced. The tumors were sliced into 2 × 2 × 2 mm 3 fragments in sterile dishes containing RPMI 1640. Typically, each fragment was implanted into a subcutaneous area in the right anks. After one week, the mice were treated with 5 mg/kg RP215 or mIgG as the control by injection around the tumor once every two days. The tumor size was measured every other day, with calipers, and the tumor volume was calculated as follows: (width 2 × length)/2. 3.7. Sequencing and analysis of rearranged genes.
The procedures were described previously [18], The repertoire of the cancer-derived Ig V genes was compared with that of published B-cell-derived Ig V genes [8].

ChIP assay
Chromatin-IP protocols were adapted from previously reported methods [15]. Brie y, cells were crosslinked with 1% formaldehyde at room temperature for 10 minutes and the reaction was quenched by 0.125 M glycine in PBS for 5 minutes at room temperature. Cells were then washed with ice-cold PBS twice, and incubated with cell lysis buffer and nuclear lysis buffe. Chromatin was sonicated and fragmented to a size of 200-500 bp, precleared with protein G beads, and incubated with 3-5 mg of anti-AR antibodies overnight. Protein-DNA complexes were precipitated, washed, and eluted. Finally, immunoprecipitated DNA fragments were extracted by DNA extraction kits (Magen) and subjected to Q-RT-PCR. The ChIP-PCR primers used are listed in Supplementary Table 3.

Luciferase assay
The pGL3-Basic plasmid (Promega) was used as a backbone for luciferase reporter construction. Brie y, the CIgG promoter region was cloned by PCR with primers listed in Supplementary Table 3. For the dual luciferase reporter assay, 293T cells were seeded into 24-well culture plates (50000 cells/well) in triplicate for each experimental condition. A total of 0.625 µg of luciferase reporter and 62.5 ng of pRL-TK vector (Promega) with or without 0.625 µg of pEnter-AR were transfected together by using Lipofectamine 3000 (Thermo Fisher). A dual luciferase reporter assay was performed using the Dual-Luciferase Reporter Assay System (Promega) following the manufacturer's instructions. Luciferase activity was measured using the Microplate Luminometer Reader (Veritas Model #9100-002). Transfection e ciency was normalized to Renilla luciferase activity.

Statistical analyses
Statistical analyses were conducted using the SPSS 22.0 package or GraphPad Prism 7.0. Data are presented as the mean ± standard error of the mean (SEM). The CIgG expression between groups was analyzed with a χ2 test. A Student's t-test was used for comparisons between two groups. Differences in cell proliferation distributions were analyzed using a two-way analysis of variance (ANOVA) test. p < 0.05 was considered statistically signi cant.

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 signi cantly 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 pro le 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 signi cantly 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 a nity, 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 in uence 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 signi cantly reduced tumor volme and weights compared with those in mice injected with prostate cancer cells carrying the empty vector ( Fig. 2G-J).

Cigg Cells Displayed More Csc-like Characteristics
To further analyze whether CIgG high cells have CSC like characteristics, we performed colony formation, sphere formation and drug-resistance assays to determine their proliferation, self renewal and drugresistance abilities in vitro. PC3 cells with CIgG high expression displayed signi cantly higher colony forming and sphere forming e ciency and greater resistance to paclitaxel than CIgG −/low cells (Fig. 3A-C).
To con rm that CIgG high cells have tumor initiating abilities, CIgG high and CIgG −/low cells puri ed from PC3 cells were used to perform tumorigenicity assays in NOD/SCID mice. As few as 500 puri ed CIgG high 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 CIgG high mice. Moreover, tumor volume and weight in the CIgG −/low group were lower than those in the CIgG high group (Fig. 3D-E).

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 signi cantly 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 con rm 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 signi cantly reduced by cotransfection with the AR plasmid (Fig. 4K).

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 [21], 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 rst 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 con rmed with TCGA prostate cancer datasets (Fig. 5D). We next examined whether CIgG abundance is upregulated by SOX2 in prostate cancer cell lines. Using SOX2speci c 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 ndings 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 positive related 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 signi cant 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 signi cantly 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).

Discussion
In prostate cancer, resistance to hormone deprivation therapy is currently the major hurdle in treatment. Next-generation ARPIs (androgen receptor pathway inhibitors), such as ENZ and ABI have marginal e cacy [25], which is usually accompanied by reactivation of AR signaling or shifting of the phenotypes to anaplastic and NE carcinomas that are devoid of AR activity [26]. In efforts to develop more effective therapies, it is critical to improve the understanding of the molecular mechanisms underlying the development of CRPC.
SOX2 is a well-de ned transcription factor that supports the proliferation and invasiveness of prostate cancer [27], A previous study suggested that AR directly represses SOX2 in an AR-positive castrationresistant prostate cancer cell line [21]; in addition, our data con rmed this nding in a castration-sensitive prostate cancer cell line (Fig. 5C); this indicates that, that genes elevated only in CRPC are mainly involved in the aggressive growth of CRPC, However, the expression of potential CRPC driver genes elevated not only sustains CRPC but also initiates CRPC, as known CRPC driver genes are typically: (1) be upregulated in CRPC, (2) show elevated expression prior to, and during, the progression from hormonenaive prostate cancer (HNPC) to CRPC; and (3) are functionally essential for CRPC development [16], We obtained evidence that SOX2-CIgG ful lls the criteria above for CRPC drivers.
Elevated SOX2 expression can be induced due to loss of AR-mediated repression during castration. Herein, SOX2 was shown to promote castration-resistance, lineage plasticity and NE differentiation in prostate cancer [22,28,29]. We hypothesize that the ADT-induced SOX2-CIgG pathway is involved in the therapeutic resistance and progression of prostate cancer. These ndings suggest that induction of CIgG is associated with increased malignancy in vitro and in vivo and neuroendocrine marker expression (NSE), suggesting that CIgG might act as a vital regulator in CRPC development. Data from human specimens highlighted the clinical relevance of CIgG in aggressive prostate cancer tumors (Fig. 1C-F), This result is consistent with previous ndings that CIgG gene expression in prostate cancer is related to cell differentiation and clinical status [30]. We also found that CIgG activation drives malignant progression and lineage plasticity in prostate cancer by activating MAPK/ERK and AKT pathways, which have been reported to be critical for CRPC development and progression [13][14][15][16]. Thus, targeting CIgG, a potential upstream mediator of MAPK/ERK and AKT (Fig. 6), with its monoclonal antibody RP215 may improve the current treatment of CRPC.
Our results also con rm that CIgG could promote epithelial-mesenchymal transition (EMT) in prostate cancer cell lines, which is dependent on the activation of AKT, and MAPK/ERK. This result is consistent with previous ndings. [31,32]. In addition, SOX2 as a stem-like transcription factor could promote EMT [32,33]. Therefore, our ndings provide a molecular bridge by which SOX2 promotes the progression of prostate cancer, suggesting a critical role for CIgG in the development of CRPC (Fig. 6).

Conclusion
Our results show that a high level of CIgG is associated with ADT resistance and that RP215 can speci cally block the pro-oncogenic properties of CIgG. Thus, our study highlights the potential for the development of a new therapy for CRPC patients by providing a rationale for the use of RP215, the monoclonal antibody of CIgG, to combat therapeutic resistance.

9.Availabilith of data and materials
All data presented or analyzed in this study are included either in this article or in the additional les.

Figure 3
CIgGhigh prostate cancer cells displayed high CSC-like characteristics. Proliferation of CIgG high PC3 cells sorted by FACS was detected by clone formation assay(A). Mammospheres were generated from 10,000 CIgG high PC3 cells in suspension culture, and then counted(B). The drug resistance capacity of CIgG−/low and CIgG high PC3 cells to paclitaxel was analyzed by CCK8 assay(C). Images (D), and weights (E) of tumor xenografts in NOD/SCID mice 5 weeks after subcutaneous inoculation with CIgG−/low and CIgG high PC3 cells with 500 cells/spot. Data from the colony formation and mammosphere assays are presented as the mean±SEM from three independent experiments, (* p < 0.05; ** p < 0.01; *** p < 0.001). Western blotting(F-H). For some cultures, 10 nM AR ligand DHT with or without 5 µM ARN509, an AR inhibitor, was added for 24 h (I-J). Transcriptional inhibitory functions of AR as indicated by reduced luciferase reporter activity. Luminescence units were normalized using Renilla luciferase signal(K). Data from the qPCR, and luciferase assays are presented as the mean±SEM from three independent experiments, (* p < 0.05; ** p < 0.01; *** p < 0.001). SOX2 is positively associated with the induction of CIgG, and CIgG knockdown reduces MAPK/ERK and AKT activity in PCa cells and inhibits EMT. Spearman correlation analysis of SOX2 with AR in clinical tissue samples from TCGA prostate cancer datasets(A-B). Signi cance was determined using a two-tailed test. AR chromatin immunoprecipitation (ChIP) documents direct binding of AR to the SOX2 enhancer region and enrichment of the SOX2 promoter after AR ChIP was normalized as a percentage of total chromatin input. IgG served as a negative control. When compared to total input, AR signi cantly enriched for the SOX2 enhancer (p<0.05); Data represent three independent experiments(C). Spearman correlation analysis of CIgG with SOX2(D). Western blot analysis of relationship of AR, SOX2 and CIgG(E-F). the effects of CIgG knockdown on phosphorylated AKT, MEK and ERK levels and EMT were determined using CIgG knockdown (SiCIgG1 and SiCIgG2) and control (NC) cell lines(G-H). Babl/c nude mice bearing PC3 tumors were injected with 5 mg/kg of RP215 or mIgG every other day around the tumor.
Growth curves (J) images (I) and weight plots (K) of harvested tumors at the end of the assay. IHC staining of subcutaneous tumors with antibodies speci c for CIgG, pAKT, pERK, and vimentin in tumorbearing mice from L. Data from ChIP assays are presented as the mean±SEM from three independent experiments, (* p < 0.05; ** p < 0.01; *** p < 0.001).