A Novel ZIP4-HDAC4-VEGFA Axis in High-Grade Serous Ovarian Cancer.

We have recently identified ZIP4 as a novel cancer stem cell (CSC) marker in high-grade serous ovarian cancer (HGSOC). While it converts drug-resistance to cisplatin (CDDP), we unexpectedly found that ZIP4 induced sensitization of HGSOC cells to histone deacetylase inhibitors (HDACis). Mechanistically, ZIP4 selectively upregulated HDAC IIa HDACs, with little or no effect on HDACs in other classes. HDAC4 knockdown (KD) and LMK-235 inhibited spheroid formation in vitro and tumorigenesis in vivo, with hypoxia inducible factor-1 alpha (HIF1α) and endothelial growth factor A (VEGFA) as functional downstream mediators of HDAC4. Moreover, we found that ZIP4, HDAC4, and HIF1α were involved in regulating secreted VEGFA in HGSOC cells. Furthermore, we tested our hypothesis that co-targeting CSC via the ZIP4-HDAC4 axis and non-CSC using CDDP is necessary and highly effective by comparing the effects of ZIP4-knockout/KD, HDAC4-KD, and HDACis, in the presence or absence of CDDP on tumorigenesis in mouse models. Our results showed that the co-targeting strategy was highly effective. Finally, data from human HGSOC tissues showed that ZIP4 and HDAC4 were upregulated in a subset of recurrent tumors, justifying the clinical relevance of the study. In summary, our study provides a new mechanistic-based targeting strategy for HGSOC.

ZIP4 Is a Novel Cancer Stem Cell Marker in High-Grade Serous Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is one of the most deadly and heterogenic cancers. We have recently shown that ZIP4 (gene name SLC39A4), a zinc transporter, is functionally involved in cancer stem cell (CSC)-related cellular activities in HGSOC. Here, we identified ZIP4 as a novel CSC marker in HGSOC. Fluorescence-activated cell sorter (FACS)-sorted ZIP4+, but not ZIP4- cells, formed spheroids and displayed self-renewing and differentiation abilities. Over-expression of ZIP4 conferred drug resistance properties in vitro. ZIP4+, but not ZIP4- cells, formed tumors/ascites in vivo. We conducted limiting dilution experiments and showed that 100-200 ZIP4+ cells from both PE04 and PEA2 cells formed larger tumors than those from 100-200 ALDH+ cells in mice. Mechanistically, we found that ZIP4 was an upstream regulator of another CSC-marker, NOTCH3, in HGSOC cells. NOTCH3 was functionally involved in spheroid formation in vitro and tumorigenesis in vivo in HGSOC. Genetic compensation studies showed that NOTCH3, but not NOTCH1, was a critical downstream mediator of ZIP4. Furthermore, NOTCH3, but not NOTCH1, physically bound to ZIP4. Collectively, our data suggest that ZIP4 is a novel CSC marker and the new ZIP4-NOTCH3 axis represents important therapeutic targets in HGSOC.

Simultaneous Inhibition of MEK and Hh Signaling Reduces Pancreatic Cancer Metastasis.

Pancreatic cancer, mostly pancreatic ductal adenocarcinoma (PDAC), is one of the most lethal cancer types, with an estimated 44,330 death in 2018 in the US alone. While targeted therapies and immune checkpoint inhibitors have significantly improved treatment options for patients with lung cancer and renal cell carcinomas, little progress has been made in pancreatic cancer, with a dismal 5-year survival rate currently at ~8%. Upon diagnosis, the majority of pancreatic cancer cases (~80%) are already metastatic. Thus, identifying ways to reduce pancreatic cancer metastasis is an unmet medical need. Furthermore, pancreatic cancer is notorious resistant to chemotherapy. While Kirsten RAt Sarcoma virus oncogene (K-RAS) mutation is the major driver for pancreatic cancer, specific inhibition of RAS signaling has been very challenging, and combination therapy is thought to be promising. In this study, we report that combination of hedgehog (Hh) and Mitogen-activated Protein/Extracellular Signal-regulated Kinase Kinase (MEK) signaling inhibitors reduces pancreatic cancer metastasis in mouse models. In mouse models of pancreatic cancer metastasis using human pancreatic cancer cells, we found that Hh target gene Gli1 is up-regulated during pancreatic cancer metastasis. Specific inhibition of smoothened signaling significantly altered the gene expression profile of the tumor microenvironment but had no significant effects on cancer metastasis. By combining Hh signaling inhibitor BMS833923 with RAS downstream MEK signaling inhibitor AZD6244, we observed reduced number of metastatic nodules in several mouse models for pancreatic cancer metastasis. These two inhibitors also decreased cell proliferation significantly and reduced CD45⁺ cells (particularly Ly6G⁺CD11b⁺ cells). We demonstrated that depleting Ly6G⁺ CD11b⁺ cells is sufficient to reduce cancer cell proliferation and the number of metastatic nodules. In vitro, Ly6G⁺ CD11b⁺ cells can stimulate cancer cell proliferation, and this effect is sensitive to MEK and Hh inhibition. Our studies may help design novel therapeutic strategies to mitigate pancreatic cancer metastasis.

Changes in mRNA/protein expression and signaling pathways in in vivo passaged mouse ovarian cancer cells.

The cure rate for late stage epithelial ovarian cancer (EOC) has not significantly improved over several decades. New and more effective targets and treatment modalities are urgently needed. RNA-seq analyses of a syngeneic EOC cell pair, representing more and less aggressive tumor cells in vivo were conducted. Bioinformatics analyses of the RNA-seq data and biological signaling and function studies have identified new targets, such as ZIP4 in EOC. Many up-regulated tumor promoting signaling pathways have been identified which are mainly grouped into three cellular activities: 1) cell proliferation and apoptosis resistance; 2) cell skeleton and adhesion changes; and 3) carbohydrate metabolic reprograming. Unexpectedly, lipid metabolism has been the major down-regulated signaling pathway in the more aggressive EOC cells. In addition, we found that hypoxic responsive genes were at the center stage of regulation and detected functional changes were related to cancer stem cell-like activities. Moreover, our genetic, cellular, biochemical, and lipidomic analyses indicated that cells grown in 2D vs. 3D, or attached vs. suspended had dramatic changes. The important clinical implications of peritoneal cavity floating tumor cells are supported by the data proved in this work. Overall, the RNA-seq data provide a landscape of gene expression alterations during tumor progression.

The novel ZIP4 regulation and its role in ovarian cancer.

Our RNAseq analyses revealed that ZIP4 is a top gene up-regulated in more aggressive ovarian cancer cells. ZIP4's role in cancer stem cells has not been reported in any type of cancer. In addition, the role and regulation of ZIP4, a zinc transporter, have been studied in the context of extracellular zinc transporting. Factors other than zinc with ZIP4 regulatory effects are essentially unknown. ZIP4 expression and its regulation in epithelial ovarian cancer cells was assessed by immunoblotting, quantitative PCR, or immunohistochemistry staining in human ovarian tissues. Cancer stem cell-related activities were examined to evaluate the role of ZIP4 in human high-grade serous ovarian cancer cells in vitro and in vivo. RNAi and CRISPR techniques were used to knockdown or knockout ZIP4 and related genes. Ovarian cancer tissues overexpressed ZIP4 when compared with normal and benign tissues. ZIP4 knockout significantly reduced several cancer stem cell-related activities in EOC cells, including proliferation, anoikis-resistance, colony-formation, spheroid-formation, drug-resistance, and side-population in vitro. ZIP4-expressing side-population highly expressed known CSC markers ALDH1 and OCT4. ZIP4 knockout dramatically reduced tumorigenesis and ZIP4 overexpression increased tumorigenesis in vivo. In addition, the ZIP4-expressing side-population had the tumor initiating activity. Moreover, the oncolipid lysophosphatic acid effectively up-regulated ZIP4 expression via the nuclear receptor peroxisome proliferator-activated receptor gamma and lysophosphatic acid 's promoting effects in cancer stem cell-related activities in HGSOC cells was at least partially mediated by ZIP4 in an extracellular zinc-independent manner. Our critical data imply that ZIP4 is a new and important cancer stem cell regulator in ovarian cancer. Our data also provide an innovative interpretation for the apparent disconnection between low levels of zinc and up-regulation of ZIP4 in ovarian cancer tissues.

FOXM1 is a downstream target of LPA and YAP oncogenic signaling pathways in high grade serous ovarian cancer.

Lysophosphatidic acid (LPA), a prototypical ligand for G protein coupled receptors, and Forkhead box protein M1 (FOXM1), a transcription factor that regulates expression of a wide array of genes involved in cancer initiation and progression, are two important oncogenic signaling molecules in human epithelial ovarian cancers (EOC). We conducted in vitro mechanistic studies using pharmacological inhibitors, genetic forms of the signaling molecules, and RNAi-mediated gene knock-down to uncover the molecular mechanisms of how these two molecules interact in EOC cells. Additionally, in vivo mouse studies were performed to confirm the functional involvement of FOXM1 in EOC tumor formation and progression. We show for the first time that LPA up-regulates expression of active FOXM1 splice variants in a time- and dose-dependent manner in the human EOC cell lines OVCA433, CAOV3, and OVCAR5. Gi-PI3K-AKT and G12/13-Rho-YAP signaling pathways were both involved in the LPA receptor (LPA1-3) mediated up-regulation of FOXM1 at the transcriptional level. In addition, down-regulation of FOXM1 in CAOV3 xenografts significantly reduced tumor and ascites formation, metastasis, and expression of FOXM1 target genes involved in cell proliferation, migration, or invasion. Collectively, our data link the oncolipid LPA, the oncogene YAP, and the central regulator of cell proliferation/mutagenesis FOXM1 in EOC cells. Moreover, these results provide further support for the importance of these pathways as potential therapeutic targets in EOC.

Defective TGF-ß signaling in bone marrow-derived cells prevents hedgehog-induced skin tumors.

Hedgehog signaling in cancer cells drives changes in the tumor microenvironment that are incompletely understood. Here, we report that hedgehog-driven tumors exhibit an increase in myeloid-derived suppressor cells (MDSC) and a decrease in T cells, indicative of an immunosuppressive tumor microenvironment. This change was associated with activated TGF-ß signaling in several cell types in basal cell carcinomas. We determined that TGF-ß signaling in bone marrow-derived cells, not keratinocytes, regulates MDSC and promotes tumor development. Tgfbr2 deficiency in the bone marrow-derived cells also reduced the size of previously developed tumors in mice. We identified CCL2 as the major chemokine attracting MDSCs to tumor, whose expression was Tgfbr2-dependent, whereas its receptor CCR2 was highly expressed in MDSC population. CCL2 alone was sufficient to induce migration of MDSCs. Moreover, the CCR2 inhibitors prevented MDSC migration toward skin cells in vitro, and reduced MDSC accumulation and hedgehog signaling-driven tumor development in mice. Our results reveal a signaling network critical for hedgehog signaling in cancer cells to establish an effective immunosuppressive microenvironment during tumor development.

Cell population analyses during skin carcinogenesis.

Cancer development is a multiple-step process involving many cell types including cancer precursor cells, immune cells, fibroblasts and endothelial cells. Each type of cells undergoes signaling and functional changes during carcinogenesis. The current challenge for many cancer researchers is to dissect these changes in each cell type during the multiple-step process in vivo. In the last few years, the authors have developed a set of procedures to isolate different cell populations during skin cancer development using K14creER/R26-SmoM2(YFP) mice. The procedure is divided into 6 parts: 1) generating appropriate mice for the study (K14creER(+) and R26-SmoM2(YFP+) mice in this protocol); 2) inducing SmoM2(YFP) expression in mouse skin; 3) preparing mouse skin biopsies; 4) isolating epidermis from skin; 5) preparing single cells from epidermis; 6) labeling single cell populations for flow cytometry analysis. Generation of sufficient number of mice with the right genotype is the limiting step in this protocol, which may take up to two months. The rest of steps take a few hours to a few days. Within this protocol, we also include a section for troubleshooting. Although we focus on skin cancer, this protocol may be modified to apply for other animal models of human diseases.

A role for transcription factor STAT3 signaling in oncogene smoothened-driven carcinogenesis.

Activation of the Hedgehog (Hh) pathway is known to drive development of basal cell carcinoma and medulloblastomas and to associate with many other types of cancer, but the exact molecular mechanisms underlying the carcinogenesis process remain elusive. We discovered that skin tumors derived from epidermal expression of oncogenic Smo, SmoM2, have elevated levels of IL-11, IL-11Rα, and STAT3 phosphorylation at Tyr(705). The relevance of our data to human conditions was reflected by the fact that all human basal cell carcinomas examined have detectable STAT3 phosphorylation, mostly in keratinocytes. The functional relevance of STAT3 in Smo-mediated carcinogenesis was revealed by epidermal specific knockout of STAT3. We showed that removal of STAT3 from mouse epidermis dramatically reduced SmoM2-mediated cell proliferation, leading to a significant decrease in epidermal thickness and tumor development. We also observed a significant reduction of epidermal stem/progenitor cell population and cyclin D1 expression in mice with epidermis-specific knockout of STAT3. Our evidence indicates that STAT3 signaling activation may be mediated by the IL-11/IL-11Rα signaling axis. We showed that tumor development was reduced after induced expression of SmoM2 in IL-11Rα null mice. Similarly, neutralizing antibodies for IL-11 reduced the tumor size. In two Hh-responsive cell lines, ES14 and C3H10T1/2, we found that addition of Smo agonist purmorphamine is sufficient to induce STAT3 phosphorylation at Tyr(705), but this effect was abolished after IL-11Rα down-regulation by shRNAs. Taken together, our results support an important role of the IL-11Rα/STAT3 signaling axis for Hh signaling-mediated signaling and carcinogenesis.

Tumor shrinkage by cyclopamine tartrate through inhibiting hedgehog signaling.

The link of hedgehog (Hh) signaling activation to human cancer and synthesis of a variety of Hh signaling inhibitors raise great expectation that inhibiting Hh signaling may be effective in human cancer treatment. Cyclopamine (Cyc), an alkaloid from the Veratrum plant, is a specific natural product inhibitor of the Hh pathway that acts by targeting smoothened (SMO) protein. However, its poor solubility, acid sensitivity, and weak potency relative to other Hh antagonists prevent the clinical development of Cyc as a therapeutic agent. Here, we report properties of cyclopamine tartrate salt (CycT) and its activities in Hh signaling-mediated cancer in vitro and in vivo. Unlike Cyc, CycT is water soluble (5-10 mg/mL). The median lethal dose (LD50) of CycT was 62.5 mg/kg body weight compared to 43.5 mg/kg for Cyc, and the plasma half-life (T1/2) of CycT was not significantly different from that of Cyc. We showed that CycT had a higher inhibitory activity for Hh signaling-dependent motor neuron differentiation than did Cyc (IC50 = 50 nmol/L for CycT vs. 300 nmol/L for Cyc). We also tested the antitumor effectiveness of these Hh inhibitors using two mouse models of basal cell carcinomas (K14cre:Ptch1(neo/neo) and K14cre:SmoM2(YFP)). After topical application of CycT or Cyc daily for 21 days, we found that all CycT-treated mice had tumor shrinkage and decreased expression of Hh target genes. Taken together, we found that CycT is an effective inhibitor of Hh signaling-mediated carcinogenesis.

Role of fatty acid synthase in gemcitabine and radiation resistance of pancreatic cancers.

Human fatty acid synthase (FASN) is a homo-dimeric protein with multi-enzymatic activity responsible for the synthesis of palmitate. FASN expression has been found to be up-regulated in multiple types of human cancers and its expression correlates with poor prognosis possibly by causing treatment resistance. In this study, we tested if FASN expression is up-regulated in human pancreatic cancers and if its higher expression level in pancreatic cancers causes intrinsic resistance to gemcitabine and radiation. We found that FASN expression is significantly up-regulated in human pancreatic cancer tissues without any correlation to age, sex, race, and tumor stage. Knocking down or over-expressing FASN significantly down- or up-regulate resistance of pancreatic cancer cell lines to both gemcitabine and radiation treatments. These findings imply that the elevated FASN expression in pancreatic cancers may contribute to unsuccessful treatments of pancreatic cancers by causing intrinsic resistance to both chemotherapy and radiation therapy.

Requirement of TGFbeta signaling for SMO-mediated carcinogenesis.

Hedgehog (Hh) signaling, via the key signal transducer Smoothened (SMO) and Gli transcription factors, is essential for embryonic development and carcinogenesis. At present, the molecular mechanism of Hh signaling-mediated carcinogenesis is not completely understood. Using a mouse model (K14cre/R26SmoM2) of SMO-mediated basal cell carcinoma development, we identified TGFß2 as a major Hh-regulated gene. TGFß2 expression was high in the keratinocytes, with activated TGFß signaling (indicated by elevated expression of phosphorylated SMAD2/3) detected in both tumor and stroma. The significance of TGFß signaling for SMO function was demonstrated in two assays. Down-regulation of TGFß2 expression prevented Hh signaling-dependent osteoblast differentiation and motor neuron differentiation. Furthermore, inhibition of TGFß signaling by TGFß receptor I inhibitor SD208 significantly reduced tumor area in K14cre/R26SmoM2 mice. Tumor shrinkage in mice was associated with an increased number of lymphocytes, suggesting an immune suppression role of TGFß signaling. The relevance of our results to human cancer is reflected by the fact that human basal cell carcinomas, which almost always harbor activated Hh signaling, have activated TGFß signaling, as indicated by high levels of phosphorylated SMAD2 and SMAD3 in tumor and stroma. Together, our data indicate that TGFß signaling is critical for Hh signaling-mediated carcinogenesis.

Patterns of East Asian pig domestication, migration, and turnover revealed by modern and ancient DNA.

The establishment of agricultural economies based upon domestic animals began independently in many parts of the world and led to both increases in human population size and the migration of people carrying domestic plants and animals. The precise circumstances of the earliest phases of these events remain mysterious given their antiquity and the fact that subsequent waves of migrants have often replaced the first. Through the use of more than 1,500 modern (including 151 previously uncharacterized specimens) and 18 ancient (representing six East Asian archeological sites) pig (Sus scrofa) DNA sequences sampled across East Asia, we provide evidence for the long-term genetic continuity between modern and ancient Chinese domestic pigs. Although the Chinese case for independent pig domestication is supported by both genetic and archaeological evidence, we discuss five additional (and possibly) independent domestications of indigenous wild boar populations: one in India, three in peninsular Southeast Asia, and one off the coast of Taiwan. Collectively, we refer to these instances as "cryptic domestication," given the current lack of corroborating archaeological evidence. In addition, we demonstrate the existence of numerous populations of genetically distinct and widespread wild boar populations that have not contributed maternal genetic material to modern domestic stocks. The overall findings provide the most complete picture yet of pig evolution and domestication in East Asia, and generate testable hypotheses regarding the development and spread of early farmers in the Far East.

Analysis of Yak MUC1 Protein Polymorphisms and the Corresponding VNTR Structure.

The genotypes and protein polymorphisms of milk epithelial mucin (MUC1) were analyzed by touch-down PCR and SDS-PAGE respectively using blood samples and milk collected from 50 lactating yaks. A total of seven alleles were revealed, namely A, B, C, D, E, F, and G, and the corresponding number of repetitive units of variable number tandem repeat (VNTR) in MUC1 gene were 21, 20, 19, 18, 17, 16, and 15, respectively. Fifteen genotypes of MUC1 were observed in yaks. The genotypes of MUC1 gene matched completely to the phenotypes of milk MUC1 in each individual. This study demonstrated that the yak MUC1 exhibits abundant polymorphisms in both its gene and protein, and the polymorphisms are due to the expression of VNTR in MUC1 gene. The possible cluster of the VNTR was also discussed in different ruminants.