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PDF file - 391K, Figure S5. Knockdown of β-catenin abrogates the ability of AR79 to increase nuclear B-catenin expression in DU145 and C4-2B cell lines

PDF file - 290K, Figure S2. AR79 has no impact on p-HIPK2 expression in prostate cancer cell lines.

PDF file - 56K, DNA profiling of LNCaP, C4-2B and DU145

Supplemental Figure 1. Validation of gene for chemoresistance of prostate cancer to DTX. Supplemental Figure 2. Caliper measurements of subcutaneous tumors presented in Figure 5A. Supplemental Figure 3. Bone stromal cells decrease LXN expression and induce chemoresistance in LNCaP cells. Supplemental Figure 4. RAW cells do not reduce LXN expression or promote chemoresistance in PC-3 cells.

Supplemental Figure 1. Anti-Integrin alphaV antibody EPR16800 inhibits prostate cancer motility and invasiveness. Supplemental Figure 2. Anti-Integrin alphaV antibody EPR16800 inhibits expression of alphaV integrin and phosphorylation of FAK, Akt and ERK in LNCaP and C4-2B cells.

PDF file - 66K, Figure S1. Mouse body weights were not impacted by AR79 treatment.

Although docetaxel is the standard of care for advanced prostate cancer, most patients develop resistance to docetaxel. Therefore, elucidating the mechanism that underlies resistance to docetaxel is critical to enhance therapeutic intervention. Mining cDNA microarray from the PC-3 prostate cancer cell line and its docetaxel-resistant derivative (PC3-TxR) revealed decreased latexin (LXN) expression in the resistant cells. LXN expression was inversely correlated with taxane resistance in a panel of prostate cancer cell lines. LXN knockdown conferred docetaxel resistance to prostate cancer cells in vitro and in vivo, whereas LXN overexpression reduced docetaxel resistance in several prostate cancer cell lines. A mouse model of prostate cancer demonstrated that prostate cancer cells developed resistance to docetaxel in the bone microenvironment, but not the soft tissue microenvironment. This was associated with decreased LXN expression in prostate cancer cells in the bone microenvironment compared with the soft tissue microenvironment. It was identified that bone stromal cells decreased LXN expression through methylation and induced chemoresistance in prostate cancer in vitro. These findings reveal that a subset of prostate cancer develops docetaxel resistance through loss of LXN expression associated with methylation and that the bone microenvironment promotes this drug resistance phenotype.Implications: This study suggests that the LXN pathway should be further explored as a viable target for preventing or reversing taxane resistance in prostate cancer. Mol Cancer Res; 15(4); 457–66. ©2017 AACR.

Integrins that contain an integrin αV subunit contribute to multiple functions that promote cancer progression. The goal of this study was to determine whether abituzumab (DI17E6, EMD 525797), a humanized monoclonal antibody (mAb) against integrin αV impacts, prostate cancer progression. To evaluate this, prostate cancer cells were treated with DI17E6 and its effects on proliferation, apoptosis, cell-cycle, adhesion, detachment, migration, invasion and phosphorylation of downstream targets, including FAK, Akt, and ERK, were determined. DI17E6 promoted detachment and inhibited adhesion of prostate cancer cells to several extracellular matrix (ECM) proteins and cells found in the bone microenvironment but had no impact on cell viability, cell-cycle, and caspase-3/7 activity. DI17E6 inhibited migration and invasion of prostate cancer cells. In addition, DI7E6 decreased phosphorylation of FAK, Akt, and ERK. These results indicate that inhibition of integrin αV with DI17E6 inhibits several prometastatic phenotypes of prostate cancer cells and therefore provide a rationale for further evaluation of DI17E6 for diminishing prostate cancer progression.Implications: This work identifies that therapeutic targeting of integrins containing an αV integrin unit inhibits cancer progression and thus may be of clinical benefit. Mol Cancer Res; 15(7); 875–83. ©2017 AACR.

PDF file - 200K, Figure S4. AR79 increases the cell number of Du145luc cells in the presence or absence of ST-2 co-culture.

Due to its bone anabolic activity, methods to increase Wnt activity, such as inhibitors of dickkopf-1 and sclerostin, are being clinically explored. Glycogen synthase kinase (GSK3β) inhibits Wnt signaling by inducing β-catenin degradation, and a GSK3β inhibitor, AR79, is being evaluated as an osteoanabolic agent. However, Wnt activation has the potential to promote tumor growth; therefore, the goal of this study was to determine if AR79 has an impact on the progression of prostate cancer. Prostate cancer tumors were established in subcutaneous and bone sites of mice followed by AR79 administration, and tumor growth, β-catenin activation, proliferation, and apoptosis were assessed. Additionally, prostate cancer and osteoblast cell lines were treated with AR79, and β-catenin status, proliferation (with β-catenin knockdown in some cases), and proportion of ALDH+CD133+ stem-like cells were determined. AR79 promoted prostate cancer tumor growth, decreased phospho-β-catenin, increased total and nuclear β-catenin, and increased tumor-induced bone remodeling. Additionally, AR79 treatment decreased caspase-3 and increased Ki67 expression in tumors and increased bone formation in normal mouse tibiae. Similarly, AR79 inhibited β-catenin phosphorylation, increased nuclear β-catenin accumulation in prostate cancer and osteoblast cell lines, and increased proliferation of prostate cancer cells in vitro through β-catenin. Furthermore, AR79 increased the ALDH+CD133+ cancer stem cell–like proportion of the prostate cancer cell lines. In conclusion, AR79, while being bone anabolic, promotes prostate cancer cell growth through Wnt pathway activation.Implications: These data suggest that clinical application of pharmaceuticals that promote Wnt pathway activation should be used with caution as they may enhance tumor growth. Mol Cancer Res; 11(12); 1597–610. ©2013 AACR.

PDF file - 294K, Figure S3. AR79 increases bone formation in normal mouse tibiae.

Supplementary Table 1: Differential Gene Expression Analysis in Individual Cell Lines Supplementary Table 2: Genes and ranking based on their single cell expression with probability (Q value) for DU145 resistant versus sensitive cells. Supplementary Table 3: PANTHER Results for Cellular Location for Individual Cell Lines Supplementary Table 4: PANTHER Results for Molecular Function for Individual Cell Lines Supplementary Table 5: IPA Gene Pathway Results Supplementary Table 6: IPA Regulator Results

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Purpose: Cabozantinib, an orally available multityrosine kinase inhibitor with activity against mesenchymal epithelial transition factor (MET) and VEGF receptor 2 (VEGFR2), induces resolution of bone scan lesions in men with castration-resistant prostate cancer bone metastases. The purpose of this study was to determine whether cabozantinib elicited a direct antitumor effect, an indirect effect through modulating bone, or both.Experimental Design: Using human prostate cancer xenograft studies in mice, we determined the impact of cabozantinib on tumor growth in soft tissue and bone. In vitro studies with cabozantinib were performed using (i) prostate cancer cell lines to evaluate its impact on cell growth, invasive ability, and MET and (ii) osteoblast cell lines to evaluate its impact on viability and differentiation and VEGFR2.Results: Cabozantinib inhibited progression of multiple prostate cancer cell lines (Ace-1, C4-2B, and LuCaP 35) in bone metastatic and soft tissue murine models of prostate cancer, except for PC-3 prostate cancer cells in which it inhibited only subcutaneous growth. Cabozantinib directly inhibited prostate cancer cell viability and induced apoptosis in vitro and in vivo and inhibited cell invasion in vitro. Cabozantinib had a dose-dependent biphasic effect on osteoblast activity and inhibitory effect on osteoclast production in vitro that was reflected in vivo. It blocked MET and VEGFR2 phosphorylation in prostate cancer cells and osteoblast-like cells, respectively.Conclusion: These data indicate that cabozantinib has direct antitumor activity, and that its ability to modulate osteoblast activity may contribute to its antitumor efficacy. Clin Cancer Res; 20(3); 617–30. ©2013 AACR.

Supplemental Figure 1. Genetic knockdown of Notch-1 inhibits growth and docetaxel chemoresistance of prostate cancer cells similar to PF-03084014.

CCR Translation for This Article from Cabozantinib Inhibits Prostate Cancer Growth and Prevents Tumor-Induced Bone Lesions

Supplemental Figure 2. PF-03084014 combined with docetaxel suppresses tumor cell proliferation, induces apoptosis and inhibits tumor angiogenesis in mice.

PDF file 173K, Impact of cabozantinib on MET, phospho-MET, VEGFR2 and phospho-VEGFR2 expression of subcutaneous LuCaP 35 and intratibial C4-2B tumors in mice

PDF file 114K, Impact of cabozantinib on angiogenesis and CD45+ myeloid cell infiltration of intratibial and soft tissue tumors in mice

Supplemental Figure Legends

Supplementary Figures S1-S3 from Fyn Is Downstream of the HGF/MET Signaling Axis and Affects Cellular Shape and Tropism in PC3 Cells

PDF file 56K, Cabozantinib increases osteoblast perimeter and decreases osteoclast perimeter in non-tumor bone

PDF file 88K, Supplementary Figure Legends

PDF file 183K, Impact of cabozantinib on MET, phospho-MET, VEGFR2 and phospho-VEGFR2 expression of subcutaneous and intratibial PC-3 tumors in mice

Supplementary Video S2 from Fyn Is Downstream of the HGF/MET Signaling Axis and Affects Cellular Shape and Tropism in PC3 Cells

Purpose: Fyn is a member of the Src family of kinases that we have previously shown to be overexpressed in prostate cancer. This study defines the biological impact of Fyn inhibition in cancer using a PC3 prostate cancer model.Experimental Design: Fyn expression was suppressed in PC3 cells using an shRNA against Fyn (PC3/FYN-). Knockdown cells were characterized using standard growth curves and time-lapse video microscopy of wound assays and Dunn Chamber assays. Tissue microarray analysis was used to verify the physiologic relevance of the HGF/MET axis in human samples. Flank injections of nude mice were performed to assess in vivo growth characteristics.Results: HGF was found to be sufficient to drive Fyn-mediated events. Compared to control transductants (PC3/Ctrl), PC3/FYN- showed a 21% decrease in growth at 4 days (P = 0.05). PC3/FYN- cells were 34% longer than control cells (P = 0.018) with 50% increase in overall surface area (P < 0.001). Furthermore, when placed in a gradient of HGF, PC3/FYN- cells showed impaired directed chemotaxis down an HGF gradient in comparison to PC3/Ctrl (P = 0.001) despite a 41% increase in cellular movement speed. In vivo studies showed 66% difference of PC3/FYN- cell growth at 8 weeks using bidimensional measurements (P = 0.002).Conclusions: Fyn plays an important role in prostate cancer biology by facilitating cellular growth and by regulating directed chemotaxis—a key component of metastasis. This finding bears particular translational importance when studying the effect of Fyn inhibition in human subjects. Clin Cancer Res; 17(10); 3112–22. ©2011 AACR.

PDF file 97K, Cabozantinib inhibits the progression of C4-2B PCa cells in bone in vivo

PDF file 95K, Cabozantinib does not inhibit progression of PC-3luc PCa cells in bone in vivo

Purpose: To investigate the efficacy and mechanisms of Notch signaling inhibition as an adjuvant to docetaxel in castration-resistant prostate cancer (CRPC) using a γ-secretase inhibitor (GSI), PF-03084014.Experimental Design: The effect of PF-03084014 on response to docetaxel was evaluated in docetaxel-sensitive and docetaxel-resistant CRPC cell lines in vitro and in murine models. Both soft tissue and bone sites were evaluated in vivo. Impacts on cell proliferation, apoptosis, cancer stem cells, and angiogenesis were evaluated.Results: The combination of PF-03084014 plus docetaxel reduced both docetaxel-sensitive and docetaxel-resistant CRPC tumor growth in soft tissue and bone greater than either agent alone. Antitumor activity was associated with PF-03084014–induced inhibition of Notch pathway signaling; decreased survival signals (cyclin E; MEK/ERK, PI3K/AKT, EGFR and NF-κB pathway; BCL-2, BCL-XL); increased apoptotic signals (BAK, BAX; cleaved caspase-3); reduced microvessel density; reduced epithelial–mesenchymal transition; and reduced cancer stem–like cells in the tumor.Conclusions: These results reveal that PF-03084014 enhances docetaxel-mediated tumor response and provides a rationale to explore GSIs as adjunct therapy in conjunction with docetaxel for men with CRPC. Clin Cancer Res; 21(20); 4619–29. ©2015 AACR.See related commentary by Zhang and Armstrong, p. 4505

Supplementary Figure 2 from Prostate Cancer Induces Bone Metastasis through Wnt-Induced Bone Morphogenetic Protein-Dependent and Independent Mechanisms

Supplementary Figure 5: MMPs promote PCa migration and invasion. A) CM was isolated from MLO-Y4 and PCa cell lines, normalized by total protein concentration, and compared by zymography for MMP expression. Densitometry of zymograph assays is also presented. B) Densitometry of zymograph in Figure 4A of MMP activity. C) mRNA from MLO-Y4 cells exposed to pressure for 24 hours was analyzed by real time PCR for MMP2 and MMP9 expression and normalized to GAPDH. D) MLO-Y4 CM (0 mmHg) was incubated with batimastat to inhibit OCy-derived MMP. CM with batimastat or DMSO control where utilized to determine alterations in PCa migration and invasion. E) PCa cells were incubated in the indicated CM and recombinant MMP2 added at the indicated concentrations. MMP2 concentrations from 40 mmHg CM were recapitulated in 0 mmHg CM through addition of recombinant (exogenous) MMP2. Invasion of PCa towards CM with additional MMP2 was measured. PCR, densitometry, and MMP2 addition experiments were analyzed using One-way ANOVA with Bonferroni post-test. Student's two-tailed t-test was used to analyze MMP inhibition experiments. All bars represent significant relationships of p>0.05.

Supplemental Figure 1: PC3 growth in bone does not increase ImP. PC3 tumor cells were injected into SCID mice 24 hours prior to implant with a wireless pressure monitoring device. A) A small increase in ImP during week 2 was observed, but was not statistically significant. B) Significant bone lysis due to tumor growth was observed. Tumor induced bone lysis may be a mechanism for relieving the pressure associated with more osteoblastic PCa tumors such as ACE1 and DU145.

The generation of an ‘angiogenic switch’ is essential for tumor growth, yet its regulation is poorly understood. In this investigation, we explored the linkage between metastasis and angiogenesis through CXCL12/CXCR4 signaling. We found that CXCR4 regulates the expression and secretion of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1). Overexpression of PGK1 reduced the secretion of vascular endothelial growth factor and interleukin-8 and increased the generation of angiostatin. At metastatic sites, however, high levels of CXCL12 signaling through CXCR4 reduced PGK1 expression, releasing the angiogenic response for metastastic growth. These data suggest that PGK1 is a critical downstream target of the chemokine axis and an important regulator of an ‘angiogenic switch’ that is essential for tumor and metastatic growth. [Cancer Res 2007;67(1):149–59]

Supplementary Figure 3: Osteocyte conditioned media promotes viability of breast and lung cancer cell lines. OCy CM was incubated with breast (MDA-MB-231 and MCF7) and lung (A549 and H441) tumor cells for 24 hours before determination of viability using resazurin. Student's two-tailed unpaired t-test was used to assess statistical differences; p

Supplementary Figure 1 from A Glycolytic Mechanism Regulating an Angiogenic Switch in Prostate Cancer

Supplementary Figure 6: CCL5 promotes MMP transcription in OCy. MLO-Y4 cells were plated and incubated for 24 hours to produce CM. Recombinant CCL5 was then incubated with MLO-Y4 for 5 hours prior to harvesting of mRNA. Real-time PCR was performed to investigate changes in MMP expression. One-way ANOVA with Bonferroni post-test was used for analysis. All bars represent significant relationships of p>0.05.

Supplementary Figure 2: MLO-Y4 CM promotes PCa viability, migration, and invasion. CM was isolated from MLO-Y4 cells. Viability of PCa cells after 24 hour incubation with MLO-Y4 CM as measured by resazurin. Student's two-tailed unpaired t-test was used to assess statistical differences; p

Prostate cancer (PCa) is frequently accompanied by osteosclerotic (i.e., excessive bone production) bone metastases. Although bone morphogenetic proteins (BMP) and Wnts are mediators of PCa-induced osteoblastic activity, the relation between them in PCa bone metastases is unknown. The goal of this study was to define this relationship. Wnt3a and Wnt5a administration or knockdown of DKK-1, a Wnt inhibitor, induced BMP-4 and 6 expression and promoter activation in PCa cells. DKK-1 blocked Wnt activation of the BMP promoters. Transfection of C4-2B cells with axin, an inhibitor of canonical Wnt signaling, blocked Wnt3a but not Wnt5a induction of the BMP promoters. In contrast, Jnk inhibitor I blocked Wnt5a but not Wnt3a induction of the BMP promoters. Wnt3a, Wnt5a, and conditioned medium (CM) from C4-2B or LuCaP23.1 cells induced osteoblast differentiation in vitro. The addition of DKK-1 and Noggin, a BMP inhibitor, to CM diminished PCa CM–induced osteoblast differentiation in a synergistic fashion. However, pretreatment of PCa cells with DKK-1 before collecting CM blocked osteoblast differentiation, whereas pretreatment with Noggin only partially reduced osteoblast differentiation, and pretreatment with both DKK-1 and Noggin had no greater effect than pretreatment with DKK-1 alone. Additionally, knockdown of BMP expression in C4-2B cells inhibited Wnt-induced osteoblastic activity. These results show that PCa promotes osteoblast differentiation through canonical and noncanonical Wnt signaling pathways that stimulate both BMP-dependent and BMP-independent osteoblast differentiation. These results show a clear link between Wnts and BMPs in PCa-induced osteoblast differentiation and provide novel targets, including the noncanonical Wnt pathway, for therapy of PCa. [Cancer Res 2008;68(14):5785–94]

Supplementary Figure 1 from Prostate Cancer Induces Bone Metastasis through Wnt-Induced Bone Morphogenetic Protein-Dependent and Independent Mechanisms

Supplementary Figure Legends 1-4 from Prostate Cancer Induces Bone Metastasis through Wnt-Induced Bone Morphogenetic Protein-Dependent and Independent Mechanisms

Supplementary Figure 3 from Prostate Cancer Induces Bone Metastasis through Wnt-Induced Bone Morphogenetic Protein-Dependent and Independent Mechanisms

Supplementary Table 1: Pressurized MLO-Y4 secretes numerous cytokines and chemokines. CM was pressurized at 40 mmHg and compared to 0 mmHg CM by commercially available cytokine array. Table of relative densitometry values normalized to 0 mmHg CM.

Supplementary Figure 4: CCL5 promotes migration and invasion of PCa. A) CCL5 was measured in CM from non-pressurized MLO-Y4 cells after normalization by protein concentration. B) MLO-Y4 CM was incubated with neutralizing antibody (1 µg/ml) against CCL5 and isotype control antibody (1 µg/ml) before being used as a chemoattractant for PCa cell lines. Neutralization of CM derived CCL5 significantly inhibited PCa migration and invasion. C) Based upon measurements of CCL5 produced by OCy at 0 and 40 mmHg, recombinant CCL5 was added to 0 mmHg CM and used in migration and invasion assays. ELISA data was analyzed using Student's two-tailed unpaired t-test. All other data was analyzed by One-way ANOVA with Bonferroni post-test. All bars represent significant relationships of p

The majority of patients with prostate cancer treated with docetaxel develop resistance to it. To better understand the mechanism behind the acquisition of resistance, we conducted single-cell RNA-sequencing (scRNA-seq) of docetaxel-sensitive and -resistant variants of DU145 and PC3 prostate cancer cell lines. Overall, sensitive and resistant cells clustered separately. Differential gene expression analysis between resistant and sensitive cells revealed 182 differentially expressed genes common to both prostate cancer cell lines. A subset of these genes gave a gene expression profile in the resistant transcriptome-like–sensitive cells similar to the resistant cells. Exploration for functional gene pathways identified 218 common pathways between the two cell lines. Protein ubiquitination was the most differentially regulated pathway and was enriched in the resistant cells. Transcriptional regulator analysis identified 321 potential regulators across both cell lines. One of the top regulators identified was nuclear protein 1 (NUPR1). In contrast to the single-cell analysis, bulk analysis of the cells did not reveal NUPR1 as a promising candidate. Knockdown and overexpression of NUPR1 in the prostate cancer cells demonstrated that NUPR1 confers docetaxel resistance in both cell lines. Collectively, these data demonstrate the utility of scRNA-seq to identify regulators of drug resistance. Furthermore, NUPR1 was identified as a mediator of prostate cancer drug resistance, which provides the rationale to explore NUPR1 and its target genes for reversal of docetaxel resistance. Implications: Using single-cell sequencing of prostate cancer, we show that NUPR1 plays a role in docetaxel resistance.