Cell-cycle and Age-Related Modulations in Mouse Chromosome Stiffness.

The intricate structure of chromosomes is complex, and many aspects of chromosome configuration/organization remain to be fully understood. Measuring chromosome stiffness can provide valuable insights into their structure. However, the nature of chromosome stiffness, whether static or dynamic, remains elusive. In this study, we analyzed chromosome stiffness in MI and MII oocytes. We revealed that MI oocytes had a ten-fold increase in stiffness compared to mitotic chromosomes, whereas chromosome stiffness in MII oocytes was relatively low chromosome. We then investigated the contribution of meiosis-specific cohesin complexes to chromosome stiffness in MI and MII oocytes. Surprisingly, the Young's modulus of chromosomes from the three meiosis-specific cohesin mutants did not exhibit significant differences compared to the wild type, indicating that these proteins may not play a substantial role in determining chromosome stiffness. Additionally, our findings revealed an age-related increase in chromosome stiffness in MI oocytes. Age correlates with elevated DNA damage levels, so we investigated the impact of etoposide-induced DNA damage on chromosome stiffness, discovering a reduction in stiffness in response to such damage in MI oocytes. Overall, our study underscores the dynamic nature of chromosome stiffness, subject to changes influenced by the cell cycle and age.

Nanoparticle Targeting in Chemo-Resistant Ovarian Cancer Reveals Dual Axis of Therapeutic Vulnerability Involving Cholesterol Uptake and Cell Redox Balance.

Platinum (Pt)-based chemotherapy is the main treatment for ovarian cancer (OC); however, most patients develop Pt resistance (Pt-R). This work shows that Pt-R OC cells increase intracellular cholesterol through uptake via the HDL receptor, scavenger receptor type B-1 (SR-B1). SR-B1 blockade using synthetic cholesterol-poor HDL-like nanoparticles (HDL NPs) diminished cholesterol uptake leading to cell death and inhibition of tumor growth. Reduced cholesterol accumulation in cancer cells induces lipid oxidative stress through the reduction of glutathione peroxidase 4 (GPx4) leading to ferroptosis. In turn, GPx4 depletion induces decreased cholesterol uptake through SR-B1 and re-sensitizes OC cells to Pt. Mechanistically, GPx4 knockdown causes lower expression of the histone acetyltransferase EP300, leading to reduced deposition of histone H3 lysine 27 acetylation (H3K27Ac) on the sterol regulatory element binding transcription factor 2 (SREBF2) promoter and suppressing expression of this key transcription factor involved in the regulation of cholesterol metabolism. SREBF2 downregulation leads to decreased SR-B1 expression and diminished cholesterol uptake. Thus, chemoresistance and cancer cell survival under high ROS burden obligates high GPx4 and SR-B1 expression through SREBF2. Targeting SR-B1 to modulate cholesterol uptake inhibits this axis and causes ferroptosis in vitro and in vivo in Pt-R OC.

Genetic Alterations of NF-κB and Its Regulators: A Rich Platform to Advance Colorectal Cancer Diagnosis and Treatment.

Colorectal cancer (CRC) is the third leading cause of cancer mortality in the United States, with an estimated 52,000 deaths in 2023. Though significant progress has been made in both diagnosis and treatment of CRC in recent years, genetic heterogeneity of CRC-the culprit for possible CRC relapse and drug resistance, is still an insurmountable challenge. Thus, developing more effective therapeutics to overcome this challenge in new CRC treatment strategies is imperative. Genetic and epigenetic changes are well recognized to be responsible for the stepwise development of CRC malignancy. In this review, we focus on detailed genetic alteration information about the nuclear factor (NF)-κB signaling, including both NF-κB family members, and their regulators, such as protein arginine methyltransferase 5 (PRMT5), and outer dynein arm docking complex subunit 2 (ODAD2, also named armadillo repeat-containing 4, ARMC4), etc., in CRC patients. Moreover, we provide deep insight into different CRC research models, with a particular focus on patient-derived xenografts (PDX) and organoid models, and their potential applications in CRC research. Genetic alterations on NF-κB signaling components are estimated to be more than 50% of the overall genetic changes identified in CRC patients collected by cBioportal for Cancer Genomics; thus, emphasizing its paramount importance in CRC progression. Consequently, various genetic alterations on NF-κB signaling may hold great promise for novel therapeutic development in CRC. Future endeavors may focus on utilizing CRC models (e.g., PDX or organoids, or isogenic human embryonic stem cell (hESC)-derived colonic cells, or human pluripotent stem cells (hPSC)-derived colonic organoids, etc.) to further uncover the underpinning mechanism of these genetic alterations in NF-κB signaling in CRC progression. Moreover, establishing platforms for drug discovery in dishes, and developing Biobanks, etc., may further pave the way for the development of innovative personalized medicine to treat CRC in the future.

Proteolytic pan-RAS Cleavage Leads to Tumor Regression in Patient-derived Pancreatic Cancer Xenografts.

The lack of effective RAS inhibition represents a major unmet medical need in the treatment of pancreatic ductal adenocarcinoma (PDAC). Here, we investigate the anticancer activity of RRSP-DTB, an engineered biologic that cleaves the Switch I of all RAS isoforms, in KRAS-mutant PDAC cell lines and patient-derived xenografts (PDX). We first demonstrate that RRSP-DTB effectively engages RAS and impacts downstream ERK signaling in multiple KRAS-mutant PDAC cell lines inhibiting cell proliferation at picomolar concentrations. We next tested RRSP-DTB in immunodeficient mice bearing KRAS-mutant PDAC PDXs. Treatment with RRSP-DTB led to ≥95% tumor regression after 29 days. Residual tumors exhibited disrupted tissue architecture, increased fibrosis and fewer proliferating cells compared with controls. Intratumoral levels of phospho-ERK were also significantly lower, indicating in vivo target engagement. Importantly, tumors that started to regrow without RRSP-DTB shrank when treatment resumed, demonstrating resistance to RRSP-DTB had not developed. Tracking persistence of the toxin activity following intraperitoneal injection showed that RRSP-DTB is active in sera from immunocompetent mice for at least 1 hour, but absent after 16 hours, justifying use of daily dosing. Overall, we report that RRSP-DTB strongly regresses hard-to-treat KRAS-mutant PDX models of pancreatic cancer, warranting further development of this pan-RAS biologic for the management of RAS-addicted tumors.

Iodide Analogs of Arsenoplatins-Potential Drug Candidates for Triple Negative Breast Cancers.

Patients with triple negative breast cancers (TNBCs)-highly aggressive tumors that do not express estrogen, progesterone, and human epidermal growth factor 2 receptors-have limited treatment options. Fewer than 30% of women with metastatic TNBC survive five years after their diagnosis, with a mortality rate within three months after a recurrence of 75%. Although TNBCs show a higher response to platinum therapy compared to other breast cancers, drug resistance remains a major obstacle; thus, platinum drugs with novel mechanisms are urgently needed. Arsenoplatins (APs) represent a novel class of anticancer agents designed to contain the pharmacophores of the two FDA approved drugs cisplatin and arsenic trioxide (As2O3) as one molecular entity. Here, we present the syntheses, crystal structures, DFT calculations, and antiproliferative activity of iodide analogs of AP-1 and AP-2, i.e., AP-5 and AP-4, respectively. Antiproliferative studies in TNBC cell lines reveal that all AP family members are more potent than cisplatin and As2O3 alone. DFT calculations demonstrate there is a low energy barrier for hydrolysis of the platinum-halide bonds in arsenoplatins, possibly contributing to their higher cytotoxicities compared to cisplatin.

Schlafen 5 as a novel therapeutic target in pancreatic ductal adenocarcinoma.

We provide evidence that a member of the human Schlafen (SLFN) family of proteins, SLFN5, is overexpressed in human pancreatic ductal adenocarcinoma (PDAC). Targeted deletion of SLFN5 results in decreased PDAC cell proliferation and suppresses PDAC tumorigenesis in in vivo PDAC models. Importantly, high expression levels of SLFN5 correlate with worse outcomes in PDAC patients, implicating SLFN5 in the pathophysiology of PDAC that leads to poor outcomes. Our studies establish novel regulatory effects of SLFN5 on cell cycle progression through binding/blocking of the transcriptional repressor E2F7, promoting transcription of key genes that stimulate S phase progression. Together, our studies suggest an essential role for SLFN5 in PDAC and support the potential for developing new therapeutic approaches for the treatment of pancreatic cancer through SLFN5 targeting.

Single-Cells Isolation and Molecular Analysis: Focus on HER2-Low CTCs in Metastatic Breast Cancer.

Although the detection of CTCs expressing HER2 at low intensity (HER2-low CTCs) has been shown to have a negative prognostic value in metastatic breast cancer (MBC) patients, the biological intrinsic nature of HER2-low CTCs remains unexplored. Considering the technical challenges behind the selective collection of immunophenotype-specific CTCs, we developed a pipeline to individually capture HER2-low CTCs. Four different breast cancer cell lines (MDA-MB-231, T47D, MDA-MB-453, and SKBR3), that are known to express HER2 at different immunohistochemistry levels (respectively classified as 0, 1+, 2+, and 3+), were spiked in healthy donor blood tubes (7.5 mL) and processed with the CellSearch® (Menarini Silicon Biosystems, Bologna, Italy) for enrichment and the DEPArray NxT™ for single cell selection. The HER2 signal-intensities of each cell line was compared using the nonparametric Mann-Whitney U test. The optimal cut-offs to distinguish HER2 1+ from 0 and 2+ cells were calculated performing the Receiver operating characteristic (ROC) curve. Median HER2 signal-intensities detected with the DEPArray NxT™ were: 2.59 (0), 3.58 (1+), 5.23 (2+) and 38.37 (3+). DEPArray NxT efficiently differentiated each single cell line (p < 0.001). The area under the ROC curve was 0.69 and 0.70 (respectively 0 vs. 1+ and 1+ vs. 2+) and the optimal calculated cut-offs were 2.85 (lower) and 4.64 (upper). HER2-low CTCs can be detected and separately collected using predetermined intensity cut-offs. This study will allow standardized single-cell or pooled collection of HER2-low CTCs for downstream molecular analyses.

HMGA2-mediated tumorigenesis through angiogenesis in leiomyoma.

OBJECTIVE: To study the role of HMGA2 in promoting angiogenesis in uterine leiomyoma (LM). DESIGN: This study involved evaluation of vessel density and angiogenic factors in leiomyomas with HMGA2 overexpression; examining angiogenic factor expression and AKT signaling in myometrial (MM) and leiomyoma cells by introducing HMGA2 overexpression in vitro; and exploring vessel formation induced by HMGA2 overexpression both in vitro and in vivo. SETTING: University research laboratory. PATIENTS: None. INTERVENTIONS: None. MAIN OUTCOME MEASURES: The main outcome measures include vessel density in leiomyomas with HMGA2 (HMGA2-LM) or MED12 (MED12-LM) alteration; angiogenic factor expression in primary leiomyoma and in vitro cell line model; and vessel formation in leiomyoma cells with HMGA2 overexpression in vitro and in vivo. RESULTS: Angiogenic factors and receptors were significantly upregulated at mRNA and protein levels in HMGA2-LM. Specifically, HMGA2-LM exhibited increased expression of VEGFA, EGF, bFGF, TGFα, VEGFR1, and VEGFR2 compared to MED12-LM and myometrium. Overexpression of HMGA2 in MM and LM cell lines resulted in increased secretion of angiogenesis-associated factors. Secreted factors promoted human umbilical vein endothelial cell (HUVEC) migration, tube formation, and wound healing. HMGA2 overexpression upregulated IGF2BP2 and pAKT, and silencing the IGF2BP2 gene reduced pAKT levels and reduced HUVEC migration. Myometrial cells with stable HMGA2 overexpression exhibited increased colony formation and cell growth in vitro and formed xenografts with increased blood vessels. CONCLUSIONS: HMGA2-LM have a high vasculature density, which likely contributes to tumor growth and disease burden of this leiomyoma subtype. HMGA2 plays an important role in angiogenesis and the involvement of IGF2BP2-mediated pAKT activity in angiogenesis, which provides a potential novel target for therapy for this subtype of LM.

ABL1, Overexpressed in Hepatocellular Carcinomas, Regulates Expression of NOTCH1 and Promotes Development of Liver Tumors in Mice.

BACKGROUND & AIMS: We investigated whether ABL proto-oncogene 1, non-receptor tyrosine kinase (ABL1) is involved in development of hepatocellular carcinoma (HCC). METHODS: We analyzed clinical and gene expression data from The Cancer Genome Atlas. Albumin-Cre (HepWT) mice and mice with hepatocyte-specific disruption of Abl1 (HepAbl-/- mice) were given hydrodynamic injections of plasmids encoding the Sleeping Beauty transposase and transposons with the MET gene and a catenin ß1 gene with an N-terminal truncation, which induces development of liver tumors. Some mice were then gavaged with the ABL1 inhibitor nilotinib or vehicle (control) daily for 4 weeks. We knocked down ABL1 with short hairpin RNAs in Hep3B and Huh7 HCC cells and analyzed their proliferation and growth as xenograft tumors in mice. We performed RNA sequencing and gene set enrichment analysis of tumors. We knocked down or overexpressed NOTCH1 and MYC in HCC cells and analyzed proliferation. We measured levels of phosphorylated ABL1, MYC, and NOTCH1 by immunohistochemical analysis of an HCC tissue microarray. RESULTS: HCC tissues had higher levels of ABL1 than non-tumor liver tissues, which correlated with shorter survival times of patients. HepWT mice with the MET and catenin ß1 transposons developed liver tumors and survived a median 64 days; HepAbl-/- mice with these transposons developed tumors that were 50% smaller and survived a median 81 days. Knockdown of ABL1 in human HCC cells reduced proliferation, growth as xenograft tumors in mice, and expression of MYC, which reduced expression of NOTCH1. Knockdown of NOTCH1 or MYC in HCC cells significantly reduced cell growth. NOTCH1 or MYC overexpression in human HCC cells promoted proliferation and rescued the phenotype caused by ABL1 knockdown. The level of phosphorylated (activated) ABL1 correlated with levels of MYC and NOTCH1 in human HCC specimens. Nilotinib decreased expression of MYC and NOTCH1 in HCC cell lines, reduced the growth of xenograft tumors in mice, and slowed growth of liver tumors in mice with MET and catenin ß1 transposons, reducing tumor levels of MYC and NOTCH1. CONCLUSIONS: HCC samples have increased levels of ABL1 compared with nontumor liver tissues, and increased levels of ABL1 correlate with shorter survival times of patients. Loss or inhibition of ABL1 reduces proliferation of HCC cells and slows growth of liver tumors in mice. Inhibitors of ABL1 might be used for treatment of HCC.

Performance of a novel Next Generation Sequencing circulating tumor DNA (ctDNA) platform for the evaluation of samples from patients with metastatic breast cancer (MBC).

Circulating tumor DNA (ctDNA) is gaining momentum as sensitive diagnostic tool for advanced disease characterization because of its ability both to capture the tumor's heterogeneity and its dynamic adaptations. However, the consistency between all the available platforms is still debated. The aim of the study was to explore the performance of the novel diagnostic NGS platform PredicinePLUS™ and to compare its results with the clinically available Guardant360™ platform for possible analytical inconsistencies. The study suggests that PredicinePLUS™ NGS platform can detect genomic alterations and measure allele frequency in samples of MBC patients and confirmed that different NGS platforms could be potentially compared provided that certain sample management and analytical requirements are met.

Association of a novel circulating tumor DNA next-generating sequencing platform with circulating tumor cells (CTCs) and CTC clusters in metastatic breast cancer.

PURPOSE: Liquid biopsies, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs), can be used to understand disease prognosis, tumor heterogeneity, and dynamic response to treatment in metastatic breast cancer (MBC). We explored a novel, 180-gene ctDNA panel and the association of this platform with CTCs and CTC clusters. METHODS: A total of 40 samples from 22 patients with MBC were included in the study. For the primary analysis, all patients had ctDNA sequencing using the PredicinePLUS™ platform. CTCs and CTC clusters were examined using the CellSearch™ System. Clinical and pathological variables were reported using descriptive analyses. Associations between CTC count and specific genomic alterations were tested using the Mann-Whitney U test. RESULTS: Of 43 sequenced patients, 40 (93%) had at least one detectable genomic alteration with a median of 6 (range 1-22). Fifty-seven different genes were altered, and the landscape of genomic alterations was representative of MBC, including the commonly encountered alterations TP53, PTEN, PIK3CA, ATM, BRCA1, CCND1, ESR1, and MYC. In patients with predominantly hormone-receptor-positive MBC, the number of CTCs was significantly associated with alterations in ESR1 (P < 0.005), GATA3 (P < 0.05), CDH1 (P < 0.0005), and CCND1 (P < 0.05) (Mann-Whitney U test). Thirty-six percent of patients had CTC clusters, which were associated with alterations in CDH1, CCND1, and BRCA1 (all P < 0.05, Mann-Whitney U test). In an independent validation cohort, CTC enumeration confirmed significant associations with ESR1 and GATA3, while CTC clusters were significantly associated with CDH1. CONCLUSIONS: We report on a novel ctDNA platform that detected genomic alterations in the vast majority of tested patients, further indicating potential clinical utility for capturing disease heterogeneity and for disease monitoring. Detection of CTCs and CTC clusters was associated with particular genomic profiles.

Anti-inflammatory activities of Sigesbeckia glabrescens Makino: combined in vitro and in silico investigations.

BACKGROUND: Sigesbeckia glabrescens Makino (SG) is one of the important plant origins of Sigesbeckiae herba and has been widely used for the treatment of chronic inflammatory diseases in China. However, the underlying anti-inflammatory mechanism of SG is rarely investigated and reported. There are more than 40 kinds of chemical constituents in SG, but the action of the bioactive compounds of SG is still unclear. Therefore, we aimed to systemically investigate the mechanisms behind the anti-inflammatory properties of SG by combining in vitro and in silico investigations. METHODS: Cytotoxicity was measured using the 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Nitric oxide (NO) release was detected using the Griess assay. The secretion of pro-inflammatory cytokines and the expression of relevant proteins were assessed using ELISA kits and Western blots, respectively. Molecular docking was performed and scored using AutoDock via a comparison with the molecular docking of N-acetyl-d-glucosamine (NAG). RESULTS: In lipopolysaccharides (LPS)-stimulated macrophages, SG significantly inhibited NO, MCP-1, and IL-6 secretion; iNOS expression; and NF-κB activation but did not significantly affect MAPK signalling (p38, ERK, and JNK). Moreover, the results from the molecular docking prediction suggested that over 10 compounds in SG could likely target TLR4, p105, and p65. CONCLUSIONS: These findings suggest that the anti-inflammatory effects of SG are highly related to the inactivation of NF-κB. Moreover, this study provides a novel approach to investigate the effects of herbal medicine using combined in vitro and in silico investigations.

Mammalian Pum1 and Pum2 Control Body Size via Translational Regulation of the Cell Cycle Inhibitor Cdkn1b.

Body and organ size regulation in mammals involves multiple signaling pathways and remains largely enigmatic. Here, we report that Pum1 and Pum2, which encode highly conserved PUF RNA-binding proteins, regulate mouse body and organ size by post-transcriptional repression of the cell cycle inhibitor Cdkn1b. Binding of PUM1 or PUM2 to Pumilio binding elements (PBEs) in the 3' UTR of Cdkn1b inhibits translation, promoting G1-S transition and cell proliferation. Mice with null mutations in Pum1 and Pum2 exhibit gene dosage-dependent reductions in body and organ size, and deficiency for Cdkn1b partially rescues postnatal growth defects in Pum1-/- mice. We propose that coordinated tissue-specific expression of Pum1 and Pum2, which involves auto-regulatory and reciprocal post-transcriptional repression, contributes to the precise regulation of body and organ size. Hence PUM-mediated post-transcriptional control of cell cycle regulators represents an additional layer of control in the genetic regulation of organ and body size.

Human Organoids Share Structural and Genetic Features with Primary Pancreatic Adenocarcinoma Tumors.

Patient-derived pancreatic ductal adenocarcinoma (PDAC) organoid systems show great promise for understanding the biological underpinnings of disease and advancing therapeutic precision medicine. Despite the increased use of organoids, the fidelity of molecular features, genetic heterogeneity, and drug response to the tumor of origin remain important unanswered questions limiting their utility. To address this gap in knowledge, primary tumor- and patient-derived xenograft (PDX)-derived organoids, and 2D cultures for in-depth genomic and histopathologic comparisons with the primary tumor were created. Histopathologic features and PDAC representative protein markers (e.g., claudin 4 and CA19-9) showed strong concordance. DNA- and RNA-sequencing (RNAseq) of single organoids revealed patient-specific genomic and transcriptomic consistency. Single-cell RNAseq demonstrated that organoids are primarily a clonal population. In drug response assays, organoids displayed patient-specific sensitivities. In addition, the in vivo PDX response to FOLFIRINOX and gemcitabine/abraxane treatments were examined, which was recapitulated in vitro with organoids. This study has demonstrated that organoids are potentially invaluable for precision medicine as well as preclinical drug treatment studies because they maintain distinct patient phenotypes and respond differently to drug combinations and dosage. IMPLICATIONS: The patient-specific molecular and histopathologic fidelity of organoids indicate that they can be used to understand the etiology of the patient's tumor and the differential response to therapies and suggests utility for predicting drug responses.

A small molecule inhibitor of the perinucleolar compartment, ML246, attenuates growth and spread of ovarian cancer.

BACKGROUND: Ovarian cancer remains a major health problem for women as it is often diagnosed at a late stage with metastatic disease. There are limited therapeutic agents and survival rates remain poor. The perinucleolar compartment (PNC) has been shown to be associated with malignancy and is considered a surrogate phenotypic marker for metastatic cancer cells. A small molecule, ML246, was derived from a screen against PNCs. In this study, the effect of ML246 on ovarian cancer growth and spread was investigated. METHODS: SKOV3 or OVCAR3 cells were treated with ML246 in vitro and PNC was visualized with immunofluorescent staining. Cell invasion was assessed using Matrigel-coated transwell systems. SKOV3 cells were xenografted orthotopically under the ovarian bursa of immunocompromised mice. Additionally, a patient derived ovarian cancer cell line was grafted subcutaneously. Mice were treated with ML246 and tumor growth and spread was assessed. RESULTS: PNCs were prevalent in the ovarian cancer cell lines OVCAR3 and SKOV3 with higher prevalence in OVCAR3 cells. Treatment with ML246 significantly reduced PNC prevalence in OVCAR3 and SKOV3 cells. Moreover, the invasive activity of both cell lines was significantly inhibited in vitro. Orthotopic implantation of SKOV3 cells resulted in growth of the tumor on the ovary as well as spread of tumor tissues outside of the primary site on organs into the abdominal cavity. Treatment with ML246 decreased the incidence of tumors outside of the ovary. In addition, a patient-derived xenograft (PDX) line was grafted subcutaneously to monitor tumor growth. ML246 significantly attenuated growth of tumors over a 5-week treatment period. CONCLUSIONS: PNC's are present in ovarian cancer cells and treatment with ML246 decreases invasion in vitro and tumor growth and spread in vivo. Additional studies are warranted to determine the efficacy of ML246 as an inhibitor of metastatic disease in ovarian cancer and to determine its precise mechanism of action.

Pumilio2 regulates synaptic plasticity via translational repression of synaptic receptors in mice.

PUMILIO 2 (PUM2) is a member of Pumilio and FBF (PUF) family, an RNA binding protein family with phylogenetically conserved roles in germ cell development. The Drosophila Pumilio homolog is also required for dendrite morphogenesis and synaptic function via translational control of synaptic proteins, such as glutamate receptors, and recent mammalian studies demonstrated a similar role in neuronal culture with associated motor and memory abnormalities in vivo. Importantly, transgenic mice with PUM2 knockout show prominent epileptiform activity, and patients with intractable temporal lobe epilepsy and mice with pilocarpine-induced seizures have decreased neuronal PUM2, possibly leading to further seizure susceptibility. However, how PUM2 influences synaptic function in vivo and, subsequently, seizures is not known. We found that PUM2 is highly expressed in the brain, especially in the temporal lobe, and knockout of Pum2 (Pum2-/- ) resulted in significantly increased pyramidal cell dendrite spine and synapse density. In addition, multiple proteins associated with excitatory synaptic function, including glutamate receptor 2 (GLUR2), are up-regulated in Pum2-/- mice. The expression of GLUR2 protein but not mRNA is increased in the Pum2-/- mutant hippocampus, Glur2 transcripts are increased in mutant polysome fractions, and overexpression of PUM2 led to repression of reporter expression containing the 3'Untranslated Region (3'UTR) of Glur2, suggesting translation of GLUR2 was increased in the absence of Pum2. Overall, these studies provide a molecular mechanism for the increased temporal lobe excitability observed with PUM2 loss and suggest PUM2 might contribute to intractable temporal lobe epilepsy.

HE4 and eIF3a Expression Correlates with Surgical Outcome and Overall Survival in Ovarian Cancer Patients with Secondary Cytoreduction.

For recurrent ovarian cancer (ROC), secondary cytoreductive surgery (SCS) is recommended as one optional treatment. However, little is known about the expression and clinical significance of biomarkers during SCS. Human epididymis protein 4 (HE4) is a clinical biomarker for ovarian cancer. Eukaryotic translation initiation factor 3a (eIF3a) is investigated extensively as a potential biomarker for malignancy. The purpose of this study was to investigate the expressions of HE4 and eIF3a at SCS, as well as their associations with surgical outcome and survival in ROC patients. Immunohistochemistry was performed to determine the expressions of HE4 and eIF3a in ovarian tumors taken from both initial and secondary cytoreductive surgery of 35 ROC patients. eIF3a levels were significantly increased at SCS, compared to those at initial cytoreductive surgery (ICS), while HE4 levels were similar. Both HE4 and eIF3a expressions were associated with surgical outcome, in terms of residual tumor. For ICS, patients with high HE4 expression achieved a higher incidence of optimal cytoreduction than those with low HE4 expression (81.0% vs. 33.3%, P = 0.015). A similar result happened in SCS, indicated by higher incidence of no residual tumor in patients with high HE4 expression (76.4% vs. 44.4%, P = 0.046). And high HE4 expression at SCS was more likely to enhance surgical outcome of SCS (77.8% vs. 29.4%, P = 0.038). Therefore, high HE4 expression at either surgery is a predictor of better overall survival (OS) (P = 0.011 and 0.002). Furthermore, patients with an elevated total score (TS) of HE4 between the two surgeries tended to have prolonged OS, compared to those with a non-elevated TS of HE4 (P = 0.076). For eIF3a, initial eIF3a expression was associated with secondary residual tumor (P = 0.035), and the difference in eIF3a expression between the two surgeries correlated with OS (P = 0.052). The expressions of HE4 and eIF3a in tumor specimens correlated with surgical outcome and predicted OS in ROC patients with SCS, thus meriting further investigation.

Cellular kinetics of MED12-mutant uterine leiomyoma growth and regression in vivo.

Cellular mechanisms of uterine leiomyoma (LM) formation have been studied primarily utilizing in vitro models. However, recent studies established that the cells growing in the primary cultures of MED12-mutant LM (MED12-LM) do not carry causal mutations. To improve the accuracy of LM research, we addressed the cellular mechanisms of LM growth and regression utilizing a patient-derived xenograft (PDX) model, which faithfully replicates the patient tumors in situ The growth and maintenance of MED12-LMs depend on 17ß-estradiol (E2) and progesterone (P4). We determined E2 and P4-activated MAPK and PI3K pathways in PDXs with upregulation of IGF1 and IGF2, suggesting that the hormone actions on MED12-LM are mediated by the IGF pathway. When hormones were removed, MED12-LM PDXs lost approximately 60% of volume within 3 days through reduction in cell size. However, in contrast to general belief, the survival of LM cells was independent of E2 and/or P4, and apoptosis was not involved in the tumor regression. Furthermore, it was postulated that abnormal collagen fibers promote the growth of LMs. However, collagen fibers of actively growing PDXs were well aligned. The disruption of collagen fibers, as found in human LM specimens, occurred only when the volume of PDXs had grown to over 20 times the volume of unstimulated PDXs, indicating disruption is the result of growth not the cause. Hence, this study revises generally accepted theories on the growth and regression of LMs.

Lentiviral CRISPR/Cas9 nickase vector mediated BIRC5 editing inhibits epithelial to mesenchymal transition in ovarian cancer cells.

BIRC5 encodes the protein survivin, a member of the inhibitor of apoptosis family. Survivin is highly expressed in a variety of cancers but has very low expression in the corresponding normal tissues, and its expression is often associated with tumor metastasis and chemoresistance. We report that survivin was highly expressed in ovarian cancer and strongly correlated with patient overall poor survival. For the first time, we provide experimental evidence that survivin is involved in epithelial to mesenchymal transition (EMT) in ovarian cancer cells. Lentiviral CRISPR/Cas9 nickase vector mediated BIRC5 gene editing led to the inhibition of EMT by upregulating epithelial cell marker, cytokeratin 7 and downregulating mesenchymal markers: snail2, ß-catenin, and vimentin in both ovarian cancer SKOV3 and OVCAR3 cells. Consistent with this molecular approach, pharmacological treatment of ovarian cancer cells using a small molecule survivin inhibitor, YM155 also inhibited EMT in these ovarian cancer cell lines. Overexpression of BIRC5 promoted EMT in SKOV3 cells. Using molecular or pharmacological approaches, we found that cell proliferation, migration, and invasion were significantly inhibited following BIRC5 disruption in both cell lines. Inhibition of BIRC5 expression also sensitized cell responses to paclitaxel treatment. Moreover, loss of BIRC5 expression attenuated TGFß signaling in both SKOV3 and OVCAR3 cells. Collectively, our studies demonstrated that disruption of BIRC5 expression inhibited EMT by attenuating the TGFß pathway in ovarian cancer cells.

Subtype-Specific Tumor-Associated Fibroblasts Contribute to the Pathogenesis of Uterine Leiomyoma.

Recent genomic studies have identified subtypes of uterine leiomyoma (LM) with distinctive genetic alterations. Here, we report the elucidation of the biological characteristics of the two most prevalent uterine leiomyoma subtypes, MED12-mutant (MED12-LM) and HMGA2-overexpressing (HMGA2-LM) uterine leiomyomas. Because each tumor carries only one genetic alteration, both subtypes are considered to be monoclonal. Approximately 90% of cells in HMGA2-uterine leiomyoma were smooth muscle cells (SMC) with HMGA2 overexpression. In contrast, MED12-LM consisted of similar numbers of SMC and non-SMC, which were mostly tumor-associated fibroblasts (TAF). Paradoxically, TAF carried no mutations in MED12, suggesting an interaction between SMC and TAF to coordinate their growth. The higher amount of extracellular matrix in MED12-LM than HMGA2-LM was partially due to the high concentration of collagen-producing TAF. SMC growth in a xenograft assay was driven by progesterone in both uterine leiomyoma subtypes. In contrast, TAF in MED12-LM proliferated in response to estradiol, whereas progesterone had no effect. The high concentration of estrogen-responsive TAF in MED12-LM explains the inconsistent discoveries between in vivo and in vitro studies on the mitogenic effect of estrogen and raises questions regarding the accuracy of previous studies utilizing MED12-LM cell culture. In addition, the differential effects of estradiol and progesterone on these uterine leiomyoma subtypes emphasize the importance of subtypes and genotypes in designing nonsurgical therapeutic strategies for uterine leiomyoma. Cancer Res; 77(24); 6891-901. ©2017 AACR.