Short-term organoid culture for drug sensitivity testing of high-grade serous carcinoma.

ObjectiveCancer patient-derived organoids (PDOs) grow as three dimensional (3D) structures in the presence of extracellular matrix and have been found to represent the original tumor's genetic complexity. In addition, PDOs can be grown and subjected to drug sensitivity testing in a shorter time course and with lesser expense than patient-derived xenograft models. Many patients with recurrent ovarian cancer develop malignant effusions that become refractory to chemotherapy. Since these same patients often present for palliative aspiration of ascites or pleural effusions, there is a potential opportunity to obtain tumor specimens in the form of multicellular spheroids (MCS) present in malignant effusion fluids. Our objective was to develop a short duration culture of MCS from ovarian cancer malignant effusions in conditions selected to support organoid growth and use them as a platform for empirical drug sensitivity testing.MethodsIn this study, malignant effusion specimens were collected from patients with high-grade serous ovarian carcinoma (HGSOC). MCS were recovered and subjected to culture conditions designed to support organoid growth. In a subset of specimens, RNA-sequencing was performed at two time points during the short-term culture to determine changes in transcriptome in response to culture conditions. Organoid induction was also characterized in these specimens using Ki67 staining and histologic analysis. Drug sensitivity testing was performed on all specimens.ResultsOur model describes organoids formed within days of primary culture, which can recapitulate the histological features of malignant ascites fluid and can be expanded for at least 6 days. RNA-seq analysis of four patient specimens showed that within 6 days of culture, there was significant up-regulation of genes related to cellular proliferation, epithelial-mesenchymal transition, and KRAS signaling pathways. Drug sensitivity testing identified several agents with therapeutic potential.ConclusionsShort duration organoid culture of MCS from HGSOC malignant effusions can be used as a platform for empiric drug sensitivity testing. These ex vivo models may be helpful in screening new or existing therapeutic agents prior to individualized treatment options

The P72R Polymorphism in R248Q/W p53 Mutants Modifies the Mutant Effect on Epithelial to Mesenchymal Transition Phenotype and Cell Invasion via CXCL1 Expression

High-grade serous carcinoma (HGSC), the most lethal subtype of epithelial ovarian cancer (EOC), is characterized by widespread TP53 mutations (\u3e90%), most of which are missense mutations (\u3e70%). The objective of this study was to investigate differential transcriptional targets affected by a common germline P72R SNP (rs1042522) in two p53 hotspot mutants, R248Q and R248W, and identify the mechanism through which the P72R SNP affects the neomorphic properties of these mutants. Using isogenic cell line models, transcriptomic analysis, xenografts, and patient data, we found that the P72R SNP modifies the effect of p53 hotspot mutants on cellular morphology and invasion properties. Most importantly, RNA sequencing studies identified CXCL1 a critical factor that is differentially affected by P72R SNP in R248Q and R248W mutants and is responsible for differences in cellular morphology and functional properties observed in these p53 mutants. We show that the mutants with the P72 SNP promote a reversion of the EMT phenotype to epithelial characteristics, whereas its R72 counterpart promotes a mesenchymal transition via the chemokine CXCL1. These studies reveal a new role of the P72R SNP in modulating the neomorphic properties of p53 mutants via CXCL1, which has significant implications for tumor invasion and metastasis

Effect of germline polymorphisms on somatic mutations in High Grade Serous ovarian cancer

Abstract: Ovarian cancer ranks fifth in cancer related deaths among gynecological cancers. A woman\u27s risk of getting ovarian cancer during her lifetime is about 1 in 75. Her lifetime chance of dying from ovarian cancer is about 1 in 100. High-grade serous carcinoma is the most aggressive type of ovarian cancer accounting for up to 70% of the cases and is associated with late detection and poor outcomes. Currently, the treatment modalities available fail to enhance the 5 year survival rate of the patient. Consequently, there is no effective treatment modality that can specifically target ovarian cancer to increase the lifespan and quality of life of the patient. The protein p53 has many anti-cancer functions and hence is referred to as a tumor suppressor or the guardian of the genome. This protein p53 is dysfunctional in cancer, including over 96% of High-grade serous ovarian cancer (HGSOC). As a result, the protein has altered wild-type p53 activity and is said to have gone rogue due to abnormal function. In human populations, codon 72 of p53 has either the sequence CCC, which encodes amino acid proline, or CGC, which encodes amino acid arginine. This sequence variation is called a germline polymorphism. The germline polymorphism results in a structural change of p53 giving rise to variants of distinct properties. Comparative sequence analyses in non-human primates suggested that the p53-P72 is the ancestral form (present in African and Asian populations), although p53-R72 occurs at a high frequency (\u3e50%) in populations that live away from the equator (Caucasian populations). This polymorphism occurs in a region of p53, which is known to be important for the anti-cancer functions of this protein. Hence we are trying to elucidate the role that the p53 codon 72 polymorphism that is common as a germline mutation across geographical and ethnic populations could play in manipulating the effect of somatic mutations in p53 thus affecting cancer progression

Tackling a tumor suppressor gone rogue: Can we OVARcome ovarian cancer?

Abstract: Ovarian cancer ranks fifth in cancer related deaths among gynecological cancers. A woman\u27s risk of getting ovarian cancer during her lifetime is about 1 in 75. Her lifetime chance of dying from ovarian cancer is about 1 in 100. High-grade serous carcinoma is the most aggressive type of ovarian cancer accounting for up to 70% of the cases and is associated with late detection and poor outcomes. Currently, the treatment modalities available fail to enhance the 5 year survival rate of the patient. Consequently, there is no effective treatment modality that can specifically target ovarian cancer to increase the lifespan and quality of life of the patient. The protein p53 has many anti-cancer functions and hence is referred to as a tumor suppressor or the guardian of the genome. This protein p53 is dysfunctional in cancer, including over 96% of High-grade serous ovarian cancer (HGSOC). As a result, the protein has altered wild-type p53 activity and is said to have gone rogue due to abnormal function. In human populations, position 72 of p53 has either the sequence CCC, which encodes amino acid proline, or CGC, which encodes amino acid arginine. This sequence variation is called a germline polymorphism. This germline polymorphism results in a structural change of p53 giving rise to variants of distinct properties. Comparative sequence analyses in non-human primates suggested that the p53-P72 is the ancestral form (present in African and Asian populations), although p53-R72 occurs at a high frequency (\u3e50%) in populations that live away from the equator (Caucasian populations). This polymorphism occurs in a region of p53, which is known to be important for the anti-cancer functions of this protein. Hence we are trying to elucidate the role that the p53 codon 72 polymorphism that is common as a germline mutation across geographical and ethnic populations could play in manipulating the effect of somatic mutations in p53 thus affecting cancer progression. We are figuring out how genetics can alter and manipulate cancer causing mutations thus providing a good means of early detection, preventing disease progression and reducing cancer health disparities among women

Genome-scale CRISPR knockout screen identifies TIGAR as a modifier of PARP inhibitor sensitivity.

Treatment of cancer with poly (ADP-ribose) polymerase (PARP) inhibitors is currently limited to cells defective in the homologous recombination (HR) pathway. Identification of genetic targets that induce or mimic HR deficiencies will extend the clinical utility of PARP inhibitors. Here we perform a CRISPR/Cas9-based genome-scale loss-of-function screen, using the sensitivity of PARP inhibitor olaparib as a surrogate. We identify C12orf5, encoding TP53 induced glycolysis and apoptosis regulator (TIGAR), as a modifier of PARP inhibitor response. We show that TIGAR is amplified in several cancer types, and higher expression of TIGAR associates with poor overall survival in ovarian cancer. TIGAR knockdown enhances sensitivity to olaparib in cancer cells via downregulation of BRCA1 and the Fanconi anemia pathway and increases senescence of these cells by affecting metabolic pathways and increasing the cytotoxic effects of olaparib. Our results indicate TIGAR should be explored as a therapeutic target for treating cancer and extending the use of PARP inhibitors

Heterozygous mutations in valosin-containing protein (VCP) and resistance to VCP inhibitors.

In recent years, multiple studies including ours have reported on the mechanism of resistance towards valosin-containing protein (VCP) inhibitors. While all these studies reported target alterations via mutations in VCP as the primary mechanism of resistance, discrepancies persist to date regarding the zygosity of these mutations responsible for the resistance. In addition, the extent to which resistant cells harbor additional mutations in other genes is not well described. In this study, we performed global transcript analysis of the parental and previously reported VCP inhibitor (CB-5083) resistant cells and found additional mutations in the resistant cells. However, our CRISPR-Cas9 gene editing studies indicate that specific mutations in VCP are sufficient to produce resistance to CB-5083 suggesting the importance of on-target mutations in VCP for resistance. Strikingly, our analysis indicates a preexisting heterozygous frameshift mutation at codon 616 (N616fs*) in one of the VCP alleles in HCT116 cells, and we showed that this mutant allele is subjected to the nonsense-mediated decay (NMD). Accordingly, we identified a heterozygous mutation at codon 526 (L526S) in genomic DNA sequencing but a homozygous L526S mutation in complementary DNA sequencing in our independently generated CB-5083 resistant HCT116 cells, implying that the L526S mutation occurs in the allele that does not harbor the frameshift N616fs* mutation. Our results suggest the NMD as a possible mechanism for achieving the homozygosity of VCP mutant responsible for the resistance to VCP inhibitors while resolving the discrepancies among previous studies. Our results also underscore the importance of performing simultaneous genomic and complementary DNA sequencing when attributing mutational effects on the functionality particularly for an oligomer protein like VCP

Transformable nanoparticles to bypass biological barriers in cancer treatment

Nanomedicine based drug delivery platforms provide an interesting avenue to explore for the future of cancer treatment. Here we discuss the barriers for drug delivery in cancer therapeutics and how nanomaterials have been designed to bypass these blockades through stimuli responsive transformation in the most recent update. Nanomaterials that address the challenges of each step provide a promising solution for new cancer therapeutics

Heterozygous mutations in valosin-containing protein (VCP) and resistance to VCP inhibitors.

In recent years, multiple studies including ours have reported on the mechanism of resistance towards valosin-containing protein (VCP) inhibitors. While all these studies reported target alterations via mutations in VCP as the primary mechanism of resistance, discrepancies persist to date regarding the zygosity of these mutations responsible for the resistance. In addition, the extent to which resistant cells harbor additional mutations in other genes is not well described. In this study, we performed global transcript analysis of the parental and previously reported VCP inhibitor (CB-5083) resistant cells and found additional mutations in the resistant cells. However, our CRISPR-Cas9 gene editing studies indicate that specific mutations in VCP are sufficient to produce resistance to CB-5083 suggesting the importance of on-target mutations in VCP for resistance. Strikingly, our analysis indicates a preexisting heterozygous frameshift mutation at codon 616 (N616fs*) in one of the VCP alleles in HCT116 cells, and we showed that this mutant allele is subjected to the nonsense-mediated decay (NMD). Accordingly, we identified a heterozygous mutation at codon 526 (L526S) in genomic DNA sequencing but a homozygous L526S mutation in complementary DNA sequencing in our independently generated CB-5083 resistant HCT116 cells, implying that the L526S mutation occurs in the allele that does not harbor the frameshift N616fs* mutation. Our results suggest the NMD as a possible mechanism for achieving the homozygosity of VCP mutant responsible for the resistance to VCP inhibitors while resolving the discrepancies among previous studies. Our results also underscore the importance of performing simultaneous genomic and complementary DNA sequencing when attributing mutational effects on the functionality particularly for an oligomer protein like VCP