Citations

Nuclear translocation of mothers against decapentaplegic homolog 2/3 (SMAD2/3), core transcription factors of transforming growth factor β (TGF‐β) signaling, is critical for hepatic stellate cell (HSC) differentiation into metastasis‐promoting myofibroblasts. SMAD2/3 have multiple coactivators, including WW domain‐containing transcription regulator protein 1 (WWTR1 or TAZ) and p300 acetyltransferase. In the nucleus, TAZ binds to SMAD2/3 to prevent SMAD2/3 nuclear export. However, how TAZ and SMAD2/3 enter the nucleus remains poorly understood because neither contains a nuclear localization signal (NLS), an amino acid sequence tagging proteins for nuclear transport. p300 is an NLS‐containing large scaffold protein, so we hypothesized that SMAD2/3 and TAZ may undergo nuclear import through complexing with p300. Coimmunoprecipitation, immunofluorescence, and nuclear fractionation assays revealed that TGF‐β1 promoted binding of SMAD2/3 and TAZ to p300 and that p300 inactivation disrupted TGF‐β1‐mediated SMAD2/3 and TAZ nuclear accumulation. Deleting the p300 NLS blocked TGF‐β1‐induced SMAD2/3 and TAZ nuclear transport. Consistently, p300 inactivation suppressed TGF‐β1‐mediated HSC activation and transcription of genes encoding tumor‐promoting factors, such as connective tissue growth factor, Tenascin C, Periostin, platelet‐derived growth factor C, and fibroblast growth factor 2, as revealed by microarray analysis. Chromatin immunoprecipitation‐real‐time quantitative PCR showed that canonical p300‐mediated acetylation of histones also facilitated transcription in response to TGF‐β1 stimulation. Interestingly, although both TGF‐β1‐mediated and stiffness‐mediated HSC activation require p300, comparison of gene expression data sets revealed that transcriptional targets of TGF‐β1 were distinct from those of stiffness‐p300 mechanosignaling. Lastly, in tumor/HSC coinjection and intrasplenic tumor injection models, targeting p300 of activated‐HSC/myofibroblasts by C646, short hairpin RNA, or cre ‐mediated gene disruption reduced tumor and liver metastatic growth in mice. Conclusion: p300 facilitates TGF‐β1‐stimulated HSC activation by both noncanonical (cytoplasm‐to‐nucleus shuttle for SMAD2/3 and TAZ) and canonical (histone acetylation) mechanisms. p300 is an attractive target for inhibiting HSC activation and the prometastatic liver microenvironment.

Abstract Introduction: Liver microenvironment is a critical determinant for development and progression of liver metastasis. Under TGF-β stimulation, hepatic stellate cells (HSCs), which are liver specific pericytes, transdifferentiate into myofibroblasts that promote tumor implantation and growth in the liver. The regulation of this HSC activation process in the liver however remains poorly understood. In this study, we tested a hypothesis that actin binding protein vasodilator-stimulated phosphoprotein (VASP), previously implicated in focal adhesion and migration of HSCs, may regulate the TGF-β mediated HSC activation process by interacting with and regulating TGF-β receptors. Methods: VASP shRNA and siRNA were used to knockdown VASP in primary human HSCs and LX2, an immortalized HSC cell line. TGF-β activation of HSCs was quantitated by immunofluorescence staining (IF) for alpha-smooth muscle actin (α-SMA) and Western blot analysis (WB) for α-SMA, fibronectin and P-SMAD2. Total TGF-β receptor levels were determined by WB, and plasma membrane TβRII was quantitated by biotinylation of cell surface proteins approach. Protein subcellular localization was investigated by IF and protein interactions were determined by double IF and co-immunoprecipitation (IP). Regulation of stellate cell VASP by HSC/tumor interactions was investigated by IF on human colorectal liver metastases and the role of stellate cell VASP in tumor growth was studied in a tumor/HSC coimplantation mouse model. Results: TGF-β1 induced more than 60% of human primary HSCs into myofibroblasts as determined by α-SMA IF. Two different VASP shRNAs significantly reduced this effect of TGF-β1 on HSC activation (P<0.05). The effect of VASP knockdown on HSC activation was confirmed in LX2 cells as well as using a VASP siRNA approach. VASP knockdown increased total TGF receptor protein levels with no effects on SMAD2 and 3 protein levels. VASP interacted with TβRII as evidenced by double IF and co-IP. Biotin-labeling of cell surface proteins and IF revealed that VASP knockdown reduced TβRII protein level at the plasma membrane and that VASP overexpression increased TβRII at the plasma membrane. Concurrently, VASP knockdown reduced plasma membrane localization of Rab11a, a small GTPase that marks recycling endosomes and is essential for recycling of plasma membrane receptors. VASP/Rab11 interactions were confirmed by IF and co-IP. Furthermore, we found that VASP knockdown HSCs significantly reduced tumor growth in mice as compared to control HSCs in a HSC/tumor coimplantation model. In patients, metastasis of colorectal cancer cells in liver sinusoids induced upregulation of VASP and α-SMA in adjacent HSCs as revealed by IF on biopsies of human colorectal liver metastases. Conclusions: Our studies demonstrate a requirement of VASP in the TGF-β dependent HSC activation in the tumor microenvironment and ensuing tumor growth. VASP interacts with Rab11 and regulates Rab11 positive recycling endosomal function thereby promoting TβRII recycling back to the plasma membrane in HSCs. VASP in the tumor microenvironment thus presents a therapeutic target for reducing tumor implantation and metastatic growth in the liver. Citation Format: Kangsheng Tu, Jiachu Li, Chunsheng Liu, Vijay H. Shah, Ningling Kang. Vasodilator-stimulated phosphoprotein promotes TGF-beta mediated myofibroblastic activation by regulating recycling of TGF-beta receptor II to the plasma membrane. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A70. doi:10.1158/1538-7445.CHTME14-A70

Platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) signaling are required for hepatic stellate cell (HSC) activation under pathological conditions such as liver metastatic tumor growth. These two signaling pathways are functionally divergent; PDGF signaling promotes proliferation and migration of HSCs, and TGF-β induces transdifferentiation of quiescent HSCs into myofibroblasts. Although PDGF signaling is implicated in TGF-β-mediated epithelial mesenchymal transition of tumor cells, the role of PDGF receptors in TGF-β activation of HSCs has not been investigated. Here we report that PDGF receptor-α (PDGFR-α) is required for TGF-β signaling of cultured human HSCs although HSCs express both PDGF-α and -β receptors. PDGFR-α knockdown inhibits TGF-β-induced phosphorylation and nuclear accumulation of SMAD2 with no influence on AKT or ERK phosphorylation associated with noncanonical TGF-β signaling. PDGFR-α knockdown suppresses TGF-β receptor I (TβRI) but increases TβRII gene transcription. At the protein level, PDGFR-α is recruited to TβRI/TβRII complexes by TGF-β stimulation. PDGFR-α knockdown blocks TGF-β-mediated internalization of TβRII and induces accumulation of TβRII at the plasma membrane, thereby inhibiting TGF-β phosphorylation of SMAD2. Functionally, knockdown of PDGFR-α reduces paracrine effects of HSCs on colorectal cancer cell proliferation and migration in vitro. In mice and patients, colorectal cancer cell invasion of the liver induces upregulation of PDGFR-α of HSCs. In summary, our finding that PDGFR-α knockdown inhibits SMAD-dependent TGF-β signaling by repressing TβRI transcriptionally and blocking endocytosis of TGF-β receptors highlights a convergence of PDGF and TGF-β signaling for HSC activation and PDGFR-α as a therapeutic target for liver metastasis and other settings of HSC activation.

Liver microenvironment is a critical determinant for development and progression of liver metastasis. Under transforming growth factor beta (TGF‐β) stimulation, hepatic stellate cells (HSCs), which are liver‐specific pericytes, transdifferentiate into tumor‐associated myofibroblasts that promote tumor implantation (TI) and growth in the liver. However, the regulation of this HSC activation process remains poorly understood. In this study, we tested whether vasodilator‐stimulated phosphoprotein (VASP) of HSCs regulated the TGF‐β‐mediated HSC activation process and tumor growth. In both an experimental liver metastasis mouse model and cancer patients, colorectal cancer cells reaching liver sinusoids induced up‐regulation of VASP and alpha‐smooth muscle actin (α‐SMA) in adjacent HSCs. VASP knockdown in HSCs inhibited TGF‐β‐mediated myofibroblastic activation of HSCs, TI, and growth in mice. Mechanistically, VASP formed protein complexes with TGF‐β receptor II (TβRII) and Rab11, a Ras‐like small GTPase and key regulator of recycling endosomes. VASP knockdown impaired Rab11 activity and Rab11‐dependent targeting of TβRII to the plasma membrane, thereby desensitizing HSCs to TGF‐β1 stimulation. Conclusions: Our study demonstrates a requirement of VASP for TGF‐β‐mediated HSC activation in the tumor microenvironment by regulating Rab11‐dependent recycling of TβRII to the plasma membrane. VASP and its effector, Rab11, in the tumor microenvironment thus present therapeutic targets for reducing TI and metastatic growth in the liver. (Hepatology 2015;61:361–374)

Abstract In the 45 years since its development, the pyrimidine analog 5-fluorouracil (5-FU) has become an integral component of many cancer treatments, most notably for the management of colorectal cancer. An appreciable fraction of patients who receive 5-FU suffer severe adverse toxicities, which in extreme cases may result in death. Dihydropyrimidine dehydrogenase (DPD, encoded by DPYD) rapidly degrades 85% of administered 5-FU, and as such, limits the amount of drug available for conversion into active metabolites. Clinical studies have suggested that genetic variations in DPYD increase the risk for 5-FU toxicity, however, there is not a clear consensus about which variations are relevant predictors. In the present study, DPYD variants were expressed in mammalian cells, and the enzymatic activity of expressed protein was determined relative to wild-type (WT). Relative sensitivity to 5-FU for cells expressing DPYD variations was also measured. The DPYD*2A variant (exon 14 deletion caused by IVS14+1G>A) was confirmed to be catalytically inactive. Compared with WT, two variants, S534N and C29R, showed significantly higher enzymatic activity. Cells expressing S534N were more resistant to 5-FU–mediated toxicity compared with cells expressing WT DPYD. These findings support the hypothesis that selected DPYD alleles are protective against severe 5-FU toxicity, and, as a consequence, may decrease the effectiveness of 5-FU an antitumor drug in carriers. In addition, this study shows a method that may be useful for phenotyping other genetic variations in pharmacologically relevant pathways. Cancer Res; 73(6); 1958–68. ©2013 AACR.

Abstract Catabolism of fluoropyrimidine drugs such as 5-fluorouracil (5-FU) and its prodrug capecitabine depends on an exclusive enzymatic step, dihydropyrimidine dehydrogenase (DPD), which results in drug inactivation. DPD exhibits wide interindividual variation, with partial to complete DPD deficiency occurring in approximately 3 to 5% of the general population. Patients with less pronounced, but decreased DPD activity may also be unable to efficiently inactivate 5-FU, such that this may contribute to 5-FU toxicity. Though numerous tests have been developed to phenotypically assess the DPD enzyme, they remain labor intensive and time consuming with difficulty in defining threshold values for separating DPD deficient patients from those with normal DPD enzyme activity. To date, attempts to develop genotypic assays have been complicated by the size and complex structure of the gene encoding the DPD enzyme (DPYD). Numerous sequence variants have been reported with varying frequencies across different populations. Unfortunately many DPYD variants have not been examined to determine whether they are associated with loss of function in the DPD enzyme. Current genotypic methods are limited due to the inability to rapidly assess multiple variants in a large number of samples at one time. In the current study, we have developed a rapid, high-throughput genotypic method using 6 multiplexed PCR assays covering 115 DPYD variants analyzed with the Sequenom matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Selected variants included previously identified and functionally characterized SNPs as well as newly identified mutations within the coding region from a previous mutation discovery analysis in Coriell cell lines and patient samples. Due to the potential importance of variants in the untranslated regions (UTRs), we have included known variants from the 5′- and 3′- UTRs (9 and 16 respectively) within our plexes. A total of 95 DNA samples derived from 4 colorectal cancer cell lines, 76 Coriell cell lines (40 Caucasian and 36 African American), and 7 Caucasian patients with clinically observed 5-FU toxicity and/or decreased DPD activity were utilized for initial assay testing. To assess “calling” efficiency and presence of false positives, 8 Coriell DNA samples plated in duplicate and 1 negative control were included. Our results showed that identified variants were 100% in agreement across duplicated samples, and only four variants within the 6 different plexes were removed due to PCR/extension failure. In summary, this method provides a comprehensive and rapid analysis of 111 DPYD variants within 6 different plexes with both sensitive and accurate detection suitable for high-throughput patient screening. Functional studies in a large patient population are underway (CA116964). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5451. doi:10.1158/1538-7445.AM2011-5451

AbstractPurpose: We conducted a phase I clinical trial to determine the maximum tolerated dose (MTD) of daily or twice daily vorinostat × 3 days when combined with fixed doses of 5-fluorouracil (FU) and leucovorin every 2 weeks.Experimental Design: Vorinostat doses were escalated in a standard 3 × 3 phase I design. FU/leucovorin was started on day 2 of vorinostat and consisted of leucovorin 400 mg/m2 i.v. over 2 hours followed by FU 400 mg/m2 i.v. bolus and 2,400 mg/m2 over 46 hours (sLV5FU2).Results: Forty-three patients were enrolled. Grade 3 fatigue, and hand and foot syndrome were the dose-limiting toxicities (DLT) at the 2,000 mg vorinostat once-daily dose level. Grade 3 fatigue and mucositis were DLTs at the 800 mg vorinostat twice-daily dose level. None of six patients at the 1,700 mg once daily or six patients at the 600 mg twice daily dose levels had a DLT; those dose levels represent the MTD. Twenty-one of 38 patients with FU-refractory colorectal cancer had stable disease, and one had a partial response. Vorinostat maximum serum concentrations at the MTD exceeded concentrations associated with thymidylate synthase downregulation in vitro. No pharmacokinetic interactions were noted between vorinostat and FU.Conclusions: The MTD of vorinostat in combination with sLV5FU2 is 1,700 mg orally once daily × 3 or 600 mg orally twice daily × 3 days every 2 weeks. Clinical activity in refractory colorectal cancer supports further clinical development of this combination. Clin Cancer Res; 16(14); 3786–94. ©2010 AACR.

Dihydropyrimidine dehydrogenase (DPD) is one of the factors that determine the efficacy and toxicity of 5-fluorouracil. Variations in DPD activity may result from alterations at the transcriptional level of the DPYD gene. Heterogeneity in DPYD expression has been reported, but the molecular mechanisms responsible for this remain unclear. We investigated methylation of the DPYD promoter as a mechanism for transcriptional regulation of DPYD in the RKO colorectal cancer cell line. We demonstrate that the active transcription machinery for DPYD is present in RKO cells, but promoter binding of Sp1, a transactivator of DPYD, was inhibited, which on subsequent examination was shown to be associated with dense promoter methylation. Treatment with 5-aza-2′-deoxycytidine alone or the combination of 5-aza-2′-deoxycytidine and trichostatin A induced demethylation of the promoter and markedly increased the DPYD mRNA level in RKO cells but not in unmethylated WiDr cells. Furthermore, in vitro methylation of the DPYD promoter decreased promoter activity. These data suggest an important role for methylation in DPYD suppression. The transcriptional suppression of DPYD by methylation may be responsible for the increased 5-fluorouracil sensitivity observed in some patients. This may also provide insight into the mechanism underlying the downregulation of DPYD in some colorectal cancers.