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[Preprint]. 2024 Feb 1:2024.01.30.577982.
doi: 10.1101/2024.01.30.577982.

Cooperativity of c-MYC with Krüppel-Like Factor 6 Splice Variant 1 induces phenotypic plasticity and promotes prostate cancer progression and metastasis

Sudeh Izadmehr  1   2   3 , Heriberto Fernandez-Hernandez  2 , Danica Wiredja  4 , Alexander Kirschenbaum  5 , Christine Lee-Poturalski  2 , Peyman Tavassoli  6 , Shen Yao  7 , Daniela Schlatzer  4 , Divya Hoon  2 , Analisa Difeo  8 , Alice C Levine  7 , Juan-Miguel Mosquera  6 , Matthew D Galsky  1 , Carlos Cordon-Cardo  3 , Goutham Narla  9
Affiliations

Cooperativity of c-MYC with Krüppel-Like Factor 6 Splice Variant 1 induces phenotypic plasticity and promotes prostate cancer progression and metastasis

Sudeh Izadmehr et al. bioRxiv. .

Abstract

Metastasis remains a major cause of morbidity and mortality in men with prostate cancer, and the functional impact of the genetic alterations, alone or in combination, driving metastatic disease remains incompletely understood. The proto-oncogene c-MYC, commonly deregulated in prostate cancer. Transgenic expression of c-MYC is sufficient to drive the progression to prostatic intraepithelial neoplasia and ultimately to moderately differentiated localized primary tumors, however, c-MYC-driven tumors are unable to progress through the metastatic cascade, suggesting that a "second-hit" is necessary in the milieu of aberrant c-MYC-driven signaling. Here, we identified cooperativity between c-MYC and KLF6-SV1, an oncogenic splice variant of the KLF6 gene. Transgenic mice that co-expressed KLF6-SV1 and c-MYC developed progressive and metastatic prostate cancer with a histological and molecular phenotype like human prostate cancer. Silencing c-MYC expression significantly reduced tumor burden in these mice supporting the necessity for c-MYC in tumor maintenance. Unbiased global proteomic analysis of tumors from these mice revealed significantly enriched vimentin, a dedifferentiation and pro-metastatic marker, induced by KLF6-SV1. c-MYC-positive tumors were also significantly enriched for KLF6-SV1 in human prostate cancer specimens. Our findings provide evidence that KLF6-SV1 is an enhancer of c-MYC-driven prostate cancer progression and metastasis, and a correlated genetic event in human prostate cancer with potential translational significance.

Keywords: Genetically Engineered Mouse Model; KLF6; KLF6-SV1; Krüppel-like factor; Metastasis; Oncogene; Prostate Cancer; Splice Variant; Transgenic Mouse Model; c-MYC.

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Conflict of interest statement

Conflict of Interest G.N. is an author on patent 20090325150 (KLF6 alternative splice forms and a germline KLF6 DNA Polymorphism associated with increased cancer risk) related to this work.

Figures

Figure 1.
Figure 1.. KLF6-SV1 cooperates with c-MYC to promote the progression of prostate tumorigenesis in vivo.
A, Generation of MYC-SV1 GEMM. Confirmation of germline transmission of transgenes. B, Representative gross images of the urogenital system of WT, KLF6-SV1, Hi-Myc and MYC-SV1 mice at 10 months of age (scale bar=1cm). C, Urogenital weights of WT, KLF6-SV1, Hi-Myc and MYC-SV1 mice at 10 months of age (mean ± s.d., P = 0.0032, ANOVA). D, IHC staining of c-MYC (brown) and KLF6-SV1 (green) co-expression of FFPE sections of representative Hi-Myc and MYC-SV1 mouse prostates at 10 months of age (63x magnification; scale bar=20 μm). E, Penetrance of histological phenotype of WT (n = 10), KLF6-SV1 (n = 11), Hi-Myc (n = 10) and MYC-SV1 (n = 10) at 10 months of age. F, Comparative pathological characterization of the mouse prostate. Photomicrographs of FFPE hematoxylin and eosin (H&E) stained sections of representative WT, KLF6-SV1, Hi-Myc and MYC-SV1 prostates at 10 months of age of age, and paired normal and adenocarcinoma (grade IV) of human prostate tissue at low (1.25x magnification, scale bar=2.5mm; 2.5x magnification, scale bar=500μm) and high (40x magnification; scale bar=50μm) magnification views of the prostate are shown. WT mice display healthy glands (red arrow); KLF6-SV1 mice exhibit PIN (blue arrow); Hi-Myc mice develop focally invasive well-differentiated prostate cancer (black arrow); MYC-SV1 mice develop diffuse poorly differentiated invasive prostate cancer (green arrow). G, IHC staining of E-cadherin (brown) of FFPE sections from representative GEMM prostates at 10 months of age (40x magnification; scale bar=50 μm). H, Microvessel count and representative IHC of CD31 stained vessels per field (mean ± s.d., P < 0.0001, ANOVA); I, PCNA-positive proliferating cells per field (mean ± s.d., P < 0.0001, ANOVA); J, TUNEL-positive apoptotic cells per field (mean ± s.d., P < 0.0001, ANOVA)
Figure 2.
Figure 2.. MYC-SV1 mice develop poorly differentiated tumors of luminal epithelial origin with metastatic disease.
A, Kaplan–Meier cumulative survival analysis shows a significant decrease in the lifespan of MYC-SV1 mice (n = 42, Median survival 477 days) compared with WT (n = 25, Medial survival = 824 days), KLF6-SV1 (n = 13, Median survival = 785 days), and Hi-Myc (n = 13, Median survival = 689 days) cohorts (P < 0.0001, Log-rank test). B, Schematic of WT and GEMM metastatic phenotypes. C, Photomicrographs of H&E and immunohistochemical stained sections of MYC-SV1 metastatic pancreatic lesion at low and high magnification (2x magnification, scale bar=500μm; 40x magnification, scale bar=50μm) magnification. Metastatic tumor nodules encircled by dashed lines (2x magnification, scale bar=500μm). Pancreatic metastasis was stained for AR, c-MYC and NKX3.1 to confirm prostate origin.
Figure 3.
Figure 3.. c-MYC and KLF6-SV1 cooperate and regulate cellular proliferation in vitro.
A, c-MYC mRNA in a panel of prostate cell lines of differing disease states. Asterisk (*) denotes cell lines with c-MYC amplification (mean ± s.d., P < 0.001, ANOVA). B, KLF6-SV1 mRNA in a panel of prostate cell lines of differing disease states (mean ± s.d., P = 0.0289, ANOVA). C, c-MYC and KLF6-SV1 protein expression in a panel of prostate cell lines of differing disease states (blue = benign cell lines, green = PIN cell lines, pink = tumorigenic cell lines; PC3 and PC3M cell lines with c-MYC amplification). D, qRT-PCR for c-MYC and E, KLF6-SV1 mRNA expression 48 hours after KLF6-SV1 and c-MYC siRNA-mediated knockdown in PC3 cells (mean ± s.d., P < 0.0001, ANOVA). F, qRT-PCR for c-MYC G, KLF6-SV1 mRNA expression with KLF6-SV1 and c-MYC knockdown by siRNA in PC3M cells (mean ± s.d., P < 0.0001, ANOVA). PC3 and PC3M cells transfected with indicated siRNAs normalized to the corresponding non-silencing control. H, KLF6-SV1 and c-MYC protein expression 48 hours after KLF6-SV1 and c-MYC knockdown by siRNA in PC3 cells. I, qRT-PCR for c-MYC mRNA expression; J, qRT-PCR for KLF6-SV1 mRNA; K, qRT-PCR for exogenous KLF6-SV1 mRNA of RWPE-1 cell lines stably overexpressing p-Babe-KLF6-SV1, p-Babe-c-MYC, or p-Babe-KLF6-SV1/c-MYC in RWPE-1 cells (mean ± s.d., P < 0.0001, ANOVA). RWPE-1 cells transfected with indicated plasmids normalized to the corresponding p-Babe control. L, KLF6-SV1 and c-MYC protein expression of RWPE-1 cell lines stably expressing p-Babe-KLF6-SV1, c-MYC, or KLF6-SV1/c-MYC. M, KI-67 mRNA expression in a panel of prostate cell lines of differing disease states (mean ± s.d., P = 0.1788, ANOVA). N, KI-67 mRNA expression 48 hours after KLF6-SV1 and c-MYC knockdown by siRNA in PC3 cells (mean ± s.d., P < 0.0001, ANOVA). O, Thymidine incorporation 48 hours after KLF6-SV1 and c-MYC knockdown by siRNA in PC3 cells (mean ± s.d., P < 0.0001, ANOVA). P, Cell cycle analysis of PC3 cells 48 hours after KLF6-SV1 and c-MYC knockdown by siRNA. Bars in graphs represent three biological replicates. Q, CDKN1A (p21) mRNA expression in a panel of prostate cell lines of differing disease states (mean ± s.d., P = 0.187, ANOVA). R, CDKN1A (p21) mRNA expression 48 hours after KLF6-SV1 and c-MYC knockdown by siRNA in PC3 cells (mean ± s.d., P = 0.0089, ANOVA).
Figure 4.
Figure 4.. Castration and de-induction of c-MYC on the maintenance of MYC-SV1 prostate tumors.
A, Schematic of castration experiment using the MYC-SV1 GEMM. Mice were castrated at 10 months of age (n = 5) post-tumor development and were sacrificed 3 months post-surgery. B, Urogenital weights of intact or castrated MYC-SV1 mice (mean ± s.d., P = 0.05, Student’s t-test). C, Incidence of mPIN and adenocarcinoma in intact and castrated mice. D, Gross images and micrographs of H&E of the urogenital system of all mice in the study. E, Histological evaluation of treatment phenotype incidence (%). H&E and immunohistochemical (IHC) staining of AR, c-MYC and KLF6-SV1 (10x magnification, scale bar=200μm; 40x magnification, scale bar=50μm). F, Percent of epithelial cells expressing AR (mean ± s.d., P = 0.1625, Student’s t-test). A total of more than 500 cells were counted from high-power fields. G, AR protein expression intensity. AR expression was measured on a 1+, 2+ or 3+ scoring system (0=negative, 1=weak, 2=moderate, 3=strong). Samples scored by intensity. Intensity distribution with representative images; primary tumors with total regions scored. H, IHC staining of CK5 (green) (mean ± s.d., P = 0.0017, Student’s t-test) and CK18 (brown) of intact and castrated prostate tissue (63x magnification, scale bar=20μm; 100x magnification, scale bar=10μm). I, Representative immunofluorescence staining image for alpha-smooth muscle actin (SMA) (mean ± s.d., P = 0.0151, Student’s t-test) (40x magnification, scale bar=50μm). J, Representative IHC staining image of PCNA (100x magnification, scale bar=10μm) with percent of epithelial cells expressing PCNA (mean ± s.d., P = 0.5784, Student’s t-test). A total of more than 500 cells were counted from high-power fields.
Figure 5.
Figure 5.. Proteomics analysis identifies a distinct expression signature with significant upregulation of vimentin in MYC-SV1 mouse model.
A, Proteomics analysis experimental design. B, Principal component analysis (PCA) including WT (n=5; black dots), KLF6-SV1 (n=5; blue dots), Hi-Myc (n=5; red dots) and MYC-SV1 (n=5; yellow dots) prostate tissue specimens. C, Heatmap of significantly altered proteins amongst GEMM groups normalized to WT or Hi-Myc, as indicated (P < 0.01, ANOVA). A magnified subset of the heatmap (group B) is shown to the right. D, Select enriched pathways. E, Western blot of vimentin protein expression of mouse prostates. F, IHC staining of vimentin shows increased expression of vimentin in MYC-SV1 tumors (10x, scale bar=250μm). E, MYC-SV1 lesions expressing high levels of vimentin protein expression also concurrently express AR and E-cadherin confirmed by IHC staining of consecutive FFPE tumor sections of MYC-SV1 prostate (10x magnification, scale bar=250μm). Representative areas are noted with an asterisk (*) and shown at high magnification (40x magnification, scale bar=50μm). H, IHC staining of consecutive sections of human prostate cancer for vimentin expression (10x magnification, scale bar=250μm; 40x magnification, scale bar=50μm). I, IHC staining of MYC-SV1 pancreatic metastases for vimentin (encircled by dashed lines). High and low magnification photomicrographs are shown (2x magnification, scale bar=500μm; 40x magnification, scale bar=50μm). J, IHC staining of vimentin expression in intact vs. castrated MYC-SV1 mouse prostate (10x magnification, scale bar=250μm; 40x magnification, scale bar=50μm).
Figure 6.
Figure 6.. KLF6-SV1 dependent up-regulation of vimentin.
A, Micrographs of immunoflourescence (IF) staining for vimentin (red) and E-cadherin (green) capturing single and dual-positive tumor cells (yellow). B, Western blot analysis of vimentin protein expression in MYC-SV1 and Hi-MYC prostate tumors. C, Western blot of exogenous expression of KLF6-SV1 in Myc-CaP cells for c-MYC, KLF6-SV1, vimentin and E-cadherin protein expression. D, E-cadherin mRNA in a panel of prostate cell lines of differing disease states (mean ± s.d., P = 0.0189, ANOVA). Fold change to RWPE-1 expression. Bars represent means ± s.d. of three biological replicates. E, qRT-PCR for vimentin mRNA expression in a panel of prostate cell lines of differing disease states (mean ± s.d., P = 0.0029, ANOVA). Fold change normalized to RWPE-1 expression. Error bars represent s.d. and experiment performed in triplicate. F, E-cadherin and vimentin protein expression in a panel of prostate cell lines of differing disease states. G, qRT-PCR for E-cadherin mRNA expression of stable cell lines over-expressing p-Babe-KLF6-SV1, p-Babe-c-MYC, or p-Babe-KLF6-SV1/c-MYC in RWPE-1 cells (mean ± s.d., P < 0.0001, ANOVA). Bars represent means of three biological replicates. H, qRT-PCR of vimentin mRNA expression of stable cell lines over-expressing p-Babe-KLF6-SV1, p-Babe-c-MYC, or p-Babe-KLF6-SV1/c-MYC in RWPE-1 cells (P < 0.0001). Bars represent means of three biological replicates. I, E-cadherin and vimentin protein expression in stable RWPE-1 cell lines over-expressing p-Babe-KLF6-SV1, p-Babe-c-MYC or p-Babe-KLF6-SV1/c-MYC. Bars represent means of three biological replicates. J, Schematic of KLF6-SV1/c-MYC-induced dedifferentiation.
Figure 7.
Figure 7.. KLF6-SV1 and c-MYC are correlated events in the human prostate.
A, Schematic of primary (n = 48 cores; 16 patients) and metastatic (liver) (n = 9 cores; 3 patients) specimens included in TMA for c-MYC and KLF6-SV1. B, Representative RNA ISH staining with specific probes against c-MYC or KLF6-SV1. Signals are granular and discrete red dots corresponding to individual RNA targets. C, Quantification and correlation between c-MYC and KLF6-SV1 mRNA (Pearson r = 0.59, P < 0.0001 and Spearman r = 0.71, P < 0.0001). D, Representative immunohistochemical staining with specific antibodies against c-MYC or KLF6-SV1. E, Quantification and correlation between c-MYC and KLF6-SV1 protein observed in human prostate specimens (Pearson r = 0.70, P < 0.0001 and Spearman r = 0.63, P < 0.0001).
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