A conditionally immortalized Gli1-positive kidney mesenchymal cell line models myofibroblast transition.

Glioma-associated oncogene homolog-1 (Gli1)-positive resident mesenchymal stem cell-like cells are the predominant source of kidney myofibroblasts in fibrosis, but investigating Gli1-positive myofibroblast progenitor activation is hampered by the difficulty of isolating and propagating primary cultures of these cells. Using a genetic strategy with positive and negative selection, we isolated Kidney-Gli1 (KGli1) cells that maintain expression of appropriate mesenchymal stem cell-like cell markers, respond to hedgehog pathway activation, and display robust myofibroblast differentiation upon treatment with transforming growth factor-ß (TGF-ß). Coculture of KGli1 cells with endothelium stabilizes capillary formation. Single-cell RNA sequencing (scRNA-seq) analysis during differentiation identified autocrine ligand-receptor pair upregulation and a strong focal adhesion pathway signal. This led us to test the serum response factor inhibitor CCG-203971 that potently inhibited TGF-ß-induced pericyte-to-myofibroblast transition. scRNA-seq also identified the unexpected upregulation of nerve growth factor (NGF), which we confirmed in two mouse kidney fibrosis models. The Ngf receptor Ntrk1 is expressed in tubular epithelium in vivo, suggesting a novel interstitial-to-tubule paracrine signaling axis. Thus, KGli1 cells accurately model myofibroblast activation in vitro, and the development of this cell line provides a new tool to study resident mesenchymal stem cell-like progenitors in health and disease.

Fibrotic Changes Mediating Acute Kidney Injury to Chronic Kidney Disease Transition.

End-stage renal disease (ESRD) is common, costly, and it results from progressive chronic kidney disease (CKD). ESRD claims many lives every year. It is increasingly recognized that episodes of acute kidney injury (AKI) predispose to the future development of CKD and ESRD. While our understanding of the pathophysiology of the AKI to CKD transition is improving, there are no validated therapeutic strategies to prevent this transition. In this review, we summarize the recent progress made in defining the cellular and molecular events underlying the AKI to CKD transition and highlight potential therapeutic targets and strategies to reduce the incidence of CKD following AKI.

Loss of PAX8 in high-grade serous ovarian cancer reduces cell survival despite unique modes of action in the fallopian tube and ovarian surface epithelium.

High-grade serous carcinoma (HGSC) is the most common and lethal form of ovarian cancer. PAX8 is a transcription factor expressed in fallopian tube epithelial cells and in 80-96% of HGSC tumors. The ovarian surface epithelium (OSE) only acquires PAX8 expression after malignant transformation. In this study, forced PAX8 expression in OSE cells increased proliferation and migration through upregulation of EMT factors such as N-cadherin and Fibronectin. OSE cells expressing PAX8 also had an increase in the FOXM1 pathway, but PAX8 alone was not sufficient to drive tumorigenesis. PAX8 knockdown in the oviductal epithelium cells did not decrease expression of the FOXM1 pathway and induced only a slight decrease in cell proliferation. No changes in migration, cell cycle, or apoptosis were detected after PAX8 knockdown in oviductal cells. Finally, PAX8 knockdown in HGSC cell lines resulted in increased apoptosis and decreased FOXM1 levels. The results presented here suggest that PAX8 has a cell specific role in governing proliferation and migration in nontransformed ovarian surface epithelium cells compared to the oviductal cells, but its reduction in serous cancer cell lines provides a common mechanism for reducing cell survival.

A Pimarane Diterpene and Cytotoxic Angucyclines from a Marine-Derived Micromonospora sp. in Vietnam's East Sea.

A screening of our actinomycete fraction library against the NCI-60 SKOV3 human tumor cell line led to the isolation of isopimara-2-one-3-ol-8,15-diene (1), lagumycin B (2), dehydrorabelomycin (3), phenanthroviridone (4), and WS-5995 A (5). These secondary metabolites were produced by a Micromonospora sp. isolated from sediment collected off the Cát Bà peninsula in the East Sea of Vietnam. Compound 1 is a novel Δ(8,9)-pimarane diterpene, representing one of approximately 20 actinomycete-produced diterpenes reported to date, while compound 2 is an angucycline antibiotic that has yet to receive formal characterization. The structures of 1 and 2 were elucidated by combined NMR and MS analysis and the absolute configuration of 1 was assigned by analysis of NOESY NMR and CD spectroscopic data. Compounds 2-5 exhibited varying degrees of cytotoxicity against a panel of cancerous and non-cancerous cell lines. Overall, this study highlights our collaborative efforts to discover novel biologically active molecules from the large, underexplored, and biodiversity-rich waters of Vietnam's East Sea.

Mutant p53 expression in fallopian tube epithelium drives cell migration.

Ovarian cancer is the fifth leading cause of cancer death among US women. Evidence supports the hypothesis that high-grade serous ovarian cancers (HGSC) may originate in the distal end of the fallopian tube. Although a heterogeneous disease, 96% of HGSC contain mutations in p53. In addition, the "p53 signature," or overexpression of p53 protein (usually associated with mutation), is a potential precursor lesion of fallopian tube derived HGSC suggesting an essential role for p53 mutation in early serous tumorigenesis. To further clarify p53-mutation dependent effects on cells, murine oviductal epithelial cells (MOE) were stably transfected with a construct encoding for the R273H DNA binding domain mutation in p53, the most common mutation in HGSC. Mutation in p53 was not sufficient to transform MOE cells but did significantly increase cell migration. A similar p53 mutation in murine ovarian surface epithelium (MOSE), another potential progenitor cell for serous cancer, was not sufficient to transform the cells nor change migration suggesting tissue specific effects of p53 mutation. Microarray data confirmed expression changes of pro-migratory genes in p53(R273H) MOE compared to parental cells, which could be reversed by suppressing Slug expression. Combining p53(R273H) with KRAS(G12V) activation caused transformation of MOE into high-grade sarcomatoid carcinoma when xenografted into nude mice. Elucidating the specific role of p53(R273H) in the fallopian tube will improve understanding of changes at the earliest stage of transformation. This information can help develop chemopreventative strategies to prevent the accumulation of additional mutations and reverse progression of the "p53 signature" thereby, improving survival rates.

Diazaquinomycins E-G, novel diaza-anthracene analogs from a marine-derived Streptomyces sp.

As part of our program to identify novel secondary metabolites that target drug-resistant ovarian cancers, a screening of our aquatic-derived actinomycete fraction library against a cisplatin-resistant ovarian cancer cell line (OVCAR5) led to the isolation of novel diaza-anthracene antibiotic diazaquinomycin E (DAQE; 1), the isomeric mixture of diazaquinomycin F (DAQF; 2) and diazaquinomycin G (DAQG; 3), and known analog diazaquinomycin A (DAQA; 4). The structures of DAQF and DAQG were solved through deconvolution of X-Ray diffraction data of their corresponding co-crystal. DAQE and DAQA exhibited moderate LC50 values against OVCAR5 of 9.0 and 8.8 µM, respectively. At lethal concentrations of DAQA, evidence of DNA damage was observed via induction of apoptosis through cleaved-PARP. Herein, we will discuss the isolation, structure elucidation, and biological activity of these secondary metabolites.

Mutation or loss of p53 differentially modifies TGFß action in ovarian cancer.

Ovarian cancer is the most lethal gynecological disease affecting women in the US. The Cancer Genome Atlas Network identified p53 mutations in 96% of high-grade serous ovarian carcinomas, demonstrating its critical role. Additionally, the Transforming Growth Factor Beta (TGFß) pathway is dysfunctional in various malignancies, including ovarian cancer. This study investigated how expression of wild-type, mutant, or the absence of p53 alters ovarian cancer cell response to TGFß signaling, as well as the response of the ovarian surface epithelium and the fallopian tube epithelium to TGFß. Only ovarian cancer cells expressing wild-type p53 were growth inhibited by TGFß, while ovarian cancer cells that were mutant or null p53 were not. TGFß induced migration in p53 null SKOV3 cells, which was not observed in SKOV3 cells with stable expression of mutant p53 R273H. Knockdown of wild-type p53 in the OVCA 420 ovarian cancer cells enhanced cell migration in response to TGFß. Increased protein expression of DKK1 and TMEPAI, two pro-invasive genes with enhanced expression in late stage metastatic ovarian cancer, was observed in p53 knockdown and null cells, while cells stably expressing mutant p53 demonstrated lower DKK1 and TMEPAI induction. Expression of mutant p53 or loss of p53 permit continued proliferation of ovarian cancer cell lines in the presence of TGFß; however, cells expressing mutant p53 exhibit reduced migration and decreased protein levels of DKK1 and TMEPAI.