<div>AbstractPurpose:<p>Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer type, lacking effective therapies and associated with a dismal prognosis. Palbociclib is a selective CDK4/6 inhibitor, which has been shown to suppress cell proliferation in many experimental cancer models. Recently, we demonstrated that pan-mTOR inhibitors, such as MLN0128, effectively induce apoptosis, although have limited efficacy in restraining proliferation of ICC cells. Here, we tested the hypothesis that palbociclib, due to its antproliferative properties in many cancer types, might synergize with MLN0128 to impair ICC growth.</p>Experimental Design:<p>Human ICC cell lines and the AKT/YapS127A ICC mouse model were used to test the therapeutic efficacy of palbociclib and MLN0128, either alone or in combination.</p>Results:<p>Administration of palbociclib suppressed <i>in vitro</i> ICC cell growth by inhibiting cell-cycle progression. Concomitant administration of palbociclib and MLN0128 led to a pronounced, synergistic growth constraint of ICC cell lines. Furthermore, while treatment with palbociclib or MLN0128 alone resulted in tumor growth reduction in AKT/YapS127A mice, a remarkable tumor regression was achieved when the two drugs were administered simultaneously. Mechanistically, palbociclib was found to potentiate MLN0128 mTOR inhibition activity, whereas MLN0128 prevented the upregulation of cyclin D1 induced by palbociclib treatment.</p>Conclusions:<p>Our study indicates the synergistic activity of palbociclib and MLN0128 in inhibiting ICC cell proliferation. Thus, combination of CDK4/6 and mTOR inhibitors might represent a novel, promising, and effective therapeutic approach against human ICC.</p><p><i>See related commentary by Malumbres, p. 6</i></p></div>
Abstract Intrahepatic cholangiocarcinoma (iCCA) is a form of deadly malignancy with limited treatment options. Gain-of-function mutations in KRAS are one of the most frequent mutations, found in ~25% of human CCA patients. MEK inhibitors have been developed and show some activity against solid tumors with Ras mutants. However, whether MEK inhibitors are effective against KRas mutant CCAs remains unknown. In the current study, we established a new mouse iCCA model (NICD/KRas) by expressing activated forms of Notch (NICD) and KRas (KRasV12D). We investigated the therapeutic potential of MEK inhibitors in vitro using human CCA cell lines and in vivo using KRasV12/NICD iCCA preclinical model. We found that in general, CCA cell lines with KRas mutations are more sensitive to MEK inhibitor U0126. Treatment of U0126 in KRas mutant CCA cells leads to increased apoptosis and decreased expression of pro-apoptosis gene Survivin. Furthermore, we found that treating KRasV12/NICD tumor-bearing mice with MEK inhibitor PD0325901 (PD901) resulted in stable disease. At molecular levels, PD901 efficiently inhibited p-ERK expression KRasV12/NICD tumor cells, leading to increased apoptosis. In summary, our studies demonstrate that KRasV12/NICD mice represent a novel and useful preclinical model to study KRas-driven CCA development. Our data further support the usefulness of MEK inhibitors for treatment of KRas mutant CCA in patients. Citation Format: Mingjie Dong, Xianqiong Liu, Katja Evert, Kirsten Utpatel, Shanshan Zhang, Li Che, John Gordan, Diego Calvisi, Matthias evert, Yan Liu, Xin Chen. Preclinical efficacy of MEK inhibition in a K-Ras driven cholangiocarcinoma preclinical model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A212.
Background & AimsYes-associated protein (YAP) and its paralog transcriptional co-activator with post synaptic density protein, drosophila disc large tumor suppressor and zonula occludens-1-binding motif (TAZ) are 2 co-activators downstream of Hippo tumor-suppressor cascade. Both have been implicated in the development of hepatocellular carcinoma (HCC). However, whether YAP and TAZ have distinct or overlapping functions during hepatocarcinogenesis remains unknown.MethodsExpression patterns of YAP and TAZ were analyzed in human HCC samples. The requirement of Yap and/or Taz in protein kinase B (Akt)/ neuroblastoma RAS viral oncogene homolog (NRas) -driven liver tumorigenesis was analyzed using conditional Yap, Taz, and Yap;Taz knockout mice. Transcriptional programs regulated by YAP and/or TAZ were identified via RNA sequencing.ResultsWe found that in human HCC samples, an almost ubiquitous activation of YAP or TAZ occurs, underlying their role in this tumor type. Intriguingly, 70% of HCC samples showed only nuclear YAP or TAZ immunoreactivity. In the Akt/NRas liver tumor model, where nuclear Yap and Taz can be detected readily, deletion of Yap or Taz alone only mildly delayed liver tumor development, whereas their concomitant ablation strongly inhibited tumor cell proliferation and significantly suppressed Akt/NRas-driven hepatocarcinogenesis. In HCC cell lines, silencing of either YAP or TAZ led to decreased expression of both overlapping and distinct sets of genes, with the most prominent gene signatures related to cell-cycle progression and DNA replication.ConclusionsYAP and TAZ have overlapping and distinct roles in hepatocarcinogenesis. HCCs may display unique activation of YAP or TAZ, thus relying on either YAP or TAZ for their growth. Yes-associated protein (YAP) and its paralog transcriptional co-activator with post synaptic density protein, drosophila disc large tumor suppressor and zonula occludens-1-binding motif (TAZ) are 2 co-activators downstream of Hippo tumor-suppressor cascade. Both have been implicated in the development of hepatocellular carcinoma (HCC). However, whether YAP and TAZ have distinct or overlapping functions during hepatocarcinogenesis remains unknown. Expression patterns of YAP and TAZ were analyzed in human HCC samples. The requirement of Yap and/or Taz in protein kinase B (Akt)/ neuroblastoma RAS viral oncogene homolog (NRas) -driven liver tumorigenesis was analyzed using conditional Yap, Taz, and Yap;Taz knockout mice. Transcriptional programs regulated by YAP and/or TAZ were identified via RNA sequencing. We found that in human HCC samples, an almost ubiquitous activation of YAP or TAZ occurs, underlying their role in this tumor type. Intriguingly, 70% of HCC samples showed only nuclear YAP or TAZ immunoreactivity. In the Akt/NRas liver tumor model, where nuclear Yap and Taz can be detected readily, deletion of Yap or Taz alone only mildly delayed liver tumor development, whereas their concomitant ablation strongly inhibited tumor cell proliferation and significantly suppressed Akt/NRas-driven hepatocarcinogenesis. In HCC cell lines, silencing of either YAP or TAZ led to decreased expression of both overlapping and distinct sets of genes, with the most prominent gene signatures related to cell-cycle progression and DNA replication. YAP and TAZ have overlapping and distinct roles in hepatocarcinogenesis. HCCs may display unique activation of YAP or TAZ, thus relying on either YAP or TAZ for their growth.
According to the theories of traditional Chinese medicine, spleen deficiency often leads to diarrhea, and deep-fried Atractylodis Rhizoma (DAR) is commonly used for the treatment. However, the association between spleen deficiency and diarrhea remains unclear. The present study aimed to investigate the therapeutic effect of DAR for the treatment of diarrhea caused by spleen deficiency and analyze the related mechanisms. It was found that a high dose group of an ethanolic extract of deep-fried Atractylodis Rhizoma (EEDAR-H) significantly inhibited weight loss, diarrhea, and pathological changes in colon tissue induced by rhubarb. EEDAR-H was found to significantly reduce the level of intestinal inflammatory cytokines and increase the expression of gastrointestinal motility hormones. In addition, EEDAR-H significantly increased the expression of aquaporin 3 (AQP3) and aquaporin 8 (AQP8) and restored abnormal water metabolism; Shen-Ling-Bai-Zhu-San (SLBZS) induced the same effect as EEDAR-H. Additional tests on the mechanism found that EEDAR-H and SLBZS promoted the integrity of the intestinal barrier. Both significantly increased the expression of the tight junction protein ZO-1 and Occludin, inhibited the phosphorylation of p38MAPK and MLC, and significantly reduced the expression levels of PAR-2. Analysis of the gut microbiota indicated that overall changes in its structure were reversed after treatment with EEDAR-H or SLBZS, in addition to significant modulation of the abundance of different phyla. At the genus level, EEDAR-H or SLBZS significantly reduced the levels of potential pathogens and increased those of beneficial bacteria.
<p>Supplementary Figure 1. Activation of the CDK4/6 axis in human intrahepatic cholangiocarcinoma (ICC) specimens. Supplementary Figure 2. Levels of Rb1, CDK4, CDK6, E2F1, and Cyclin E1 mRNA in human intrahepatic cholangiocarcinoma (ICC) from The Cancer Genome Atlas (TCGA) and National Cancer Institute (NCI) datasets. Supplementary Figure 3: Effect of Palbociclib on ICC cell growth. Supplementary Figure 4: Effect of Palbociclib on the levels of putative target proteins in human ICC cell lines. Supplementary Figure 5. Inhibition of CyclinD1 (CCND1) expression enhances tumor cell growth suppressor capacity of Palbobiclib. Supplementary Figure 6. Correlation analysis of Retinoblastoma (Rb) expression and the growth inhibitory effects of Palbociclib. Supplementary Figure 7. Palbociclib inhibits proliferation of ICC cell lines in colony formation assays. Supplementary Figure 8. Retinoblastoma-dependent cell growth inhibition effect of long-term Palbociclib treatment in ICC cells. Supplementary Figure 9. Effect of combined Palbociclib/MLN0128 treatment on cell proliferation. Supplementary Figure 10. Synergistic effect of Palbociclib/MLN0128 combination in ICC cell lines. Supplementary Figure 11. Effect of combined Palbociclib/MLN0128 administration on cell cycle of ICC cell lines. Supplementary Figure 12. Effect of Palbociclib/MLN0128 on putative target proteins in intrahepatic cholangiocarcinoma (ICC) cell lines. Supplementary Figure 13. Overview of the apoptosis rate in ICC lesions from AKT/YapS127A mice subjected to the various treatment regimens. Supplementary Figure 14. Effect of Palbociclib/MLN0128 treatment on putative target proteins in AKT/YapS127A mice. Supplementary Table 1: Clinicopathological features of intrahepatic cholangiocarcinoma (ICC) patients Supplementary Table 2: Western blotting antibody information</p>
Abstract Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer. It lacks effective therapies and is associated with high mortality rate as well as poor prognosis. Our recent study suggests that treatment of pan-mTOR inhibitor MLN0128 leads to stable disease in murine ICC model induced by activated AKT and Yap (AKT/YapS127A) via inducing apoptosis without affecting tumor cell proliferation. Palbociclib is a pan CDK4/6 inhibitor that has been approved by the FDA for the treatment of HR-positive advanced-stage breast cancer. However, its efficacy in ICC has not been investigated. ICC cells are known to be highly proliferative. Here we hypothesize that palbociclib is effective against ICC by inhibiting tumor cell proliferation. Furthermore, it may synergize with MLN0128 to inhibit ICC growth in vivo. We tested the therapeutic efficacy of palbociclib alone or in combination with MLN0128 in human ICC cell lines as well as in AKT/YapS127A ICC model. We found that in vitro palbociclib inhibits ICC cell growth via inhibiting cell cycle progression. The long-term cell growth, but not the short-term cell survival, to palbociclib is RB-dependent. Concomitant treatment of palbociclib with MLN0128 has a synergistic effect in inhibiting ICC cell viability. In AKT/YapS127A ICC preclinical model, treatment with palbociclib or MLN0128 alone all led to stable disease. In striking contrast, combined treatment of palbociclib with MLN0128 resulted in significant tumor regression. Mechanistically, we found that combined palbociclib with MLN0128 has a profound inhibitory effect in inhibiting ICC cell proliferation both in vivo and in vitro. In summary, our study suggests that palbociclib synergizes with MLN0128 to inhibit ICC cell proliferation, and combined treatment of palbociclib with MLN0128 represents promising novel therapeutics against this deadly malignancy. Citation Format: Xinhua Song, Xianqiong Liu, Jingxiao Wang, Xin Chen. CDK4/6 inhibitor synergizes with mTOR inhibitor to suppress cholangiocarcinoma pathogenesis [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B153.
Ferroptosis is a novel process of regulated cell death discovered in recent years, mainly caused by intracellular lipid peroxidation. It is morphologically manifested as shrinking of mitochondria, swelling of cytoplasm and organelles, rupture of plasma membrane, and formation of double-membrane vesicles. Work done in the past 5 years indicates that induction of ferroptosis is a promising strategy in the treatment of hepatocellular carcinoma (HCC). System xc - /GSH/GPX4 , iron metabolism, p53 and lipid peroxidation pathways are the main focus areas in ferroptosis research. In this paper, we analyze the ferroptosis-inducing drugs and experimental agents that have been used in the last 5 years in the treatment of HCC. We summarize four different key molecular mechanisms that induce ferroptosis, i.e., system xc - /GSH/GPX4 , iron metabolism, p53 and lipid peroxidation. Finally, we outline the prognostic analysis associated with ferroptosis in HCC. The findings summarized suggest that ferroptosis induction can serve as a promising new therapeutic approach for HCC and can provide a basis for clinical diagnosis and prevention of this disease.
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