Selective targeting of IRAK1 attenuates low molecular weight hyaluronic acid-induced stemness and non-canonical STAT3 activation in epithelial ovarian cancer.

Advanced epithelial ovarian cancer (EOC) survival rates are dishearteningly low, with ~25% surviving beyond 5 years. Evidence suggests that cancer stem cells contribute to acquired chemoresistance and tumor recurrence. Here, we show that IRAK1 is upregulated in EOC tissues, and enhanced expression correlates with poorer overall survival. Moreover, low molecular weight hyaluronic acid, which is abundant in malignant ascites from patients with advanced EOC, induced IRAK1 phosphorylation leading to STAT3 activation and enhanced spheroid formation. Knockdown of IRAK1 impaired tumor growth in peritoneal disease models, and impaired HA-induced spheroid growth and STAT3 phosphorylation. Finally, we determined that TCS2210, a known inducer of neuronal differentiation in mesenchymal stem cells, is a selective inhibitor of IRAK1. TCS2210 significantly inhibited EOC growth in vitro and in vivo both as monotherapy, and in combination with cisplatin. Collectively, these data demonstrate IRAK1 as a druggable target for EOC.

Perinucleolar Compartment (PNC) Prevalence as an Independent Prognostic Factor in Pediatric Ewing Sarcoma: A Multi-Institutional Study.

The perinucleolar compartment (PNC) is a small nuclear body that plays important role in tumorigenesis. PNC prevalence correlates with poor prognosis and cancer metastasis. Its expression in pediatric Ewing sarcoma (EWS) has not previously been documented. In this study, we analyzed 40 EWS tumor cases from Caucasian and Hispanic patients for PNC prevalence by immunohistochemical detection of polypyrimidine tract binding protein and correlated the prevalence with dysregulated microRNA profiles. EWS cases showed staining ranging from 0 to 100%, which were categorized as diffuse (≥77%, n = 9, high PNC) or not diffuse (<77%, n = 31) for low PNC. High PNC prevalence was significantly higher in Hispanic patients from the US (n = 6, p = 0.017) and in patients who relapsed with metastatic disease (n = 4; p = 0.011). High PNC was associated with significantly shorter disease-free survival and early recurrence compared to those with low PNC. Using NanoString digital profiling, high PNC tumors revealed upregulation of eight and downregulation of 18 microRNAs. Of these, miR-320d and miR-29c-3p had the most significant differential expression in tumors with high PNC. In conclusion, this is the first study that demonstrates the presence of PNC in EWS, reflecting its utility as a predictive biomarker associated with tumor metastasis, specific microRNA profile, Hispanic ethnic origin, and poor prognosis.

Anticancer Activity of Novel Difluorinated Curcumin Analog and Its Inclusion Complex with 2-Hydroxypropyl-ß-Cyclodextrin against Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the primary reason for cancer-related deaths in the US. Genetic mutations, drug resistance, the involvement of multiple signaling pathways, cancer stem cells (CSCs), and desmoplastic stroma, which hinders drug penetrance, contribute to poor chemotherapeutic efficacy. Hence, there is a need to identify novel drugs with improved delivery to improve treatment outcomes. Curcumin is one such compound that can inhibit multiple signaling pathways and CSCs. However, curcumin's clinical applicability for treating PDAC is limited because of its poor solubility in water and metabolic instability. Hence, we developed a difluorinated curcumin (CDF) analog that accumulates selectively in the pancreas and inhibits PDAC growth in vitro and in vivo. In the present work, we developed its 2-hydroxy-propyl-ß-cyclodextrin (HCD) inclusion complex to increase its water solubility and hydrolytic stability. The CDFHCD inclusion complex was characterized by spectroscopic, thermal, and microscopic techniques. The inclusion complex exhibited increased aqueous solubility, hydrolytic stability, and antiproliferative activity compared to parent CDF. Moreover, CDF and CDFHCD inhibited colony and spheroid formation, and induced cell cycle and apoptosis in PDAC cell lines. Hence, CDFHCD self-assembly is an efficient approach to increase water solubility and anticancer therapeutic efficacy, which now warrants advancement towards a clinical proof of concept in PDAC patients.

Doublecortin-like kinase 1 is a therapeutic target in squamous cell carcinoma.

Doublecortin like kinase 1 (DCLK1) plays a crucial role in several cancers including colon and pancreatic adenocarcinomas. However, its role in squamous cell carcinoma (SCC) remains unknown. To this end, we examined DCLK1 expression in head and neck SCC (HNSCC) and anal SCC (ASCC). We found that DCLK1 is elevated in patient SCC tissue, which correlated with cancer progression and poorer overall survival. Furthermore, DCLK1 expression is significantly elevated in human papilloma virus negative HNSCC, which are typically aggressive with poor responses to therapy. To understand the role of DCLK1 in tumorigenesis, we used specific shRNA to suppress DCLK1 expression. This significantly reduced tumor growth, spheroid formation, and migration of HNSCC cancer cells. To further the translational relevance of our studies, we sought to identify a selective DCLK1 inhibitor. Current attempts to target DCLK1 using pharmacologic approaches have relied on nonspecific suppression of DCLK1 kinase activity. Here, we demonstrate that DiFiD (3,5-bis [2,4-difluorobenzylidene]-4-piperidone) binds to DCLK1 with high selectivity. Moreover, DiFiD mediated suppression of DCLK1 led to G2/M arrest and apoptosis and significantly suppressed tumor growth of HNSCC xenografts and ASCC patient derived xenografts, supporting that DCLK1 is critical for SCC growth.

Mitogen-activated protein kinase-activated protein kinase-2 (MK2) and its role in cell survival, inflammatory signaling, and migration in promoting cancer.

Cancer and the immune system share an intimate relationship. Chronic inflammation increases the risk of cancer occurrence and can also drive inflammatory mediators into the tumor microenvironment enhancing tumor growth and survival. The p38 MAPK pathway is activated both acutely and chronically by stress, inflammatory chemokines, chronic inflammatory conditions, and cancer. These properties have led to extensive efforts to find effective drugs targeting p38, which have been unsuccessful. The immediate downstream serine/threonine kinase and substrate of p38 MAPK, mitogen-activated-protein-kinase-activated-protein-kinase-2 (MK2) protects cells against stressors by regulating the DNA damage response, transcription, protein and messenger RNA stability, and motility. The phosphorylation of downstream substrates by MK2 increases inflammatory cytokine production, drives an immune response, and contributes to wound healing. By binding directly to p38 MAPK, MK2 is responsible for the export of p38 MAPK from the nucleus which gives MK2 properties that make it unique among the large number of p38 MAPK substrates. Many of the substrates of both p38 MAPK and MK2 are separated between the cytosol and nucleus and interfering with MK2 and altering this intracellular translocation has implications for the actions of both p38 MAPK and MK2. The inhibition of MK2 has shown promise in combination with both chemotherapy and radiotherapy as a method for controlling cancer growth and metastasis in a variety of cancers. Whereas the current data are encouraging the field requires the development of selective and well tolerated drugs to target MK2 and a better understanding of its effects for effective clinical use.

Honokiol Affects Stem Cell Viability by Suppressing Oncogenic YAP1 Function to Inhibit Colon Tumorigenesis.

Honokiol (HNK) is a biphenolic compound that has been used in traditional medicine for treating various ailments, including cancers. In this study, we determined the effect of HNK on colon cancer cells in culture and in a colitis-associated cancer model. HNK treatment inhibited proliferation and colony formation while inducing apoptosis. In addition, HNK suppressed colonosphere formation. Molecular docking suggests that HNK interacts with reserve stem cell marker protein DCLK1, with a binding energy of -7.0 Kcal/mol. In vitro kinase assays demonstrated that HNK suppressed the DCLK1 kinase activity. HNK also suppressed the expression of additional cancer stem cell marker proteins LGR5 and CD44. The Hippo signaling pathway is active in intestinal stem cells. In the canonical pathway, YAP1 is phosphorylated at Ser127 by upstream Mst1/2 and Lats1/2. This results in the sequestration of YAP1 in the cytoplasm, thereby not allowing YAP1 to translocate to the nucleus and interact with TEAD1-4 transcription factors to induce gene expression. However, HNK suppressed Ser127 phosphorylation in YAP1, but the protein remains sequestered in the cytoplasm. We further determined that this occurs by YAP1 interacting with PUMA. To determine if this also occurs in vivo, we performed studies in an AOM/DSS induced colitis-associated cancer model. HNK administered by oral gavage at a dose of 5mg/kg bw for 24 weeks demonstrated a significant reduction in the expression of YAP1 and TEAD1 and in the stem marker proteins. Together, these data suggest that HNK prevents colon tumorigenesis in part by inducing PUMA-YAP1 interaction and cytoplasmic sequestration, thereby suppressing the oncogenic YAP1 activity.

Fosciclopirox suppresses growth of high-grade urothelial cancer by targeting the γ-secretase complex.

Ciclopirox (CPX) is an FDA-approved topical antifungal agent that has demonstrated preclinical anticancer activity in a number of solid and hematologic malignancies. Its clinical utility as an oral anticancer agent, however, is limited by poor oral bioavailability and gastrointestinal toxicity. Fosciclopirox, the phosphoryloxymethyl ester of CPX (Ciclopirox Prodrug, CPX-POM), selectively delivers the active metabolite, CPX, to the entire urinary tract following parenteral administration. We characterized the activity of CPX-POM and its major metabolites in in vitro and in vivo preclinical models of high-grade urothelial cancer. CPX inhibited cell proliferation, clonogenicity and spheroid formation, and increased cell cycle arrest at S and G0/G1 phases. Mechanistically, CPX suppressed activation of Notch signaling. Molecular modeling and cellular thermal shift assays demonstrated CPX binding to γ-secretase complex proteins Presenilin 1 and Nicastrin, which are essential for Notch activation. To establish in vivo preclinical proof of principle, we tested fosciclopirox in the validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) mouse bladder cancer model. Once-daily intraperitoneal administration of CPX-POM for four weeks at doses of 235 mg/kg and 470 mg/kg significantly decreased bladder weight, a surrogate for tumor volume, and resulted in a migration to lower stage tumors in CPX-POM treated animals. This was coupled with a reduction in the proliferation index. Additionally, there was a reduction in Presenilin 1 and Hes-1 expression in the bladder tissues of CPX-POM treated animals. Following the completion of the first-in-human Phase 1 trial (NCT03348514), the pharmacologic activity of fosciclopirox is currently being characterized in a Phase 1 expansion cohort study of muscle-invasive bladder cancer patients scheduled for cystectomy (NCT04608045) as well as a Phase 2 trial of newly diagnosed and recurrent urothelial cancer patients scheduled for transurethral resection of bladder tumors (NCT04525131).

Overcoming Wnt-ß-catenin dependent anticancer therapy resistance in leukaemia stem cells.

Leukaemia stem cells (LSCs) underlie cancer therapy resistance but targeting these cells remains difficult. The Wnt-ß-catenin and PI3K-Akt pathways cooperate to promote tumorigenesis and resistance to therapy. In a mouse model in which both pathways are activated in stem and progenitor cells, LSCs expanded under chemotherapy-induced stress. Since Akt can activate ß-catenin, inhibiting this interaction might target therapy-resistant LSCs. High-throughput screening identified doxorubicin (DXR) as an inhibitor of the Akt-ß-catenin interaction at low doses. Here we repurposed DXR as a targeted inhibitor rather than a broadly cytotoxic chemotherapy. Targeted DXR reduced Akt-activated ß-catenin levels in chemoresistant LSCs and reduced LSC tumorigenic activity. Mechanistically, ß-catenin binds multiple immune-checkpoint gene loci, and targeted DXR treatment inhibited expression of multiple immune checkpoints specifically in LSCs, including PD-L1, TIM3 and CD24. Overall, LSCs exhibit distinct properties of immune resistance that are reduced by inhibiting Akt-activated ß-catenin. These findings suggest a strategy for overcoming cancer therapy resistance and immune escape.

Suppressing STAT5 signaling affects osteosarcoma growth and stemness.

Osteosarcoma (OS) is the most common primary bone tumor that primarily affects children and adolescents. Studies suggested that dysregulation JAK/STAT signaling promotes the development of OS. Cells treated with pimozide, a STAT5 inhibitor suppressed proliferation and colony formation and induced sub G0/G1 cell cycle arrest and apoptosis. There was a reduction in cyclin D1 and CDK2 expression and Rb phosphorylation, and activation of Caspase-3 and PARP cleavage. In addition, pimozide suppressed the formation of 3-dimensional osteospheres and growth of the cells in the Tumor in a Dish lung organoid system. Furthermore, there was a reduction in expression of cancer stem cell marker proteins DCLK1, CD44, CD133, Oct-4, and ABCG2. More importantly, it was the short form of DCLK1 that was upregulated in osteospheres, which was suppressed in response to pimozide. We further confirmed by flow cytometry a reduction in DCLK1+ cells. Moreover, pimozide inhibits the phosphorylation of STAT5, STAT3, and ERK in OS cells. Molecular docking studies suggest that pimozide interacts with STAT5A and STAT5B with binding energies of -8.4 and -6.4 Kcal/mol, respectively. Binding was confirmed by cellular thermal shift assay. To further understand the role of STAT5, we knocked down the two isoforms using specific siRNAs. While knockdown of the proteins did not affect the cells, knockdown of STAT5B reduced pimozide-induced necrosis and further enhanced late apoptosis. To determine the effect of pimozide on tumor growth in vivo, we administered pimozide intraperitoneally at a dose of 10 mg/kg BW every day for 21 days in mice carrying KHOS/NP tumor xenografts. Pimozide treatment significantly suppressed xenograft growth. Western blot and immunohistochemistry analyses also demonstrated significant inhibition of stem cell marker proteins. Together, these data suggest that pimozide treatment suppresses OS growth by targeting both proliferating cells and stem cells at least in part by inhibiting the STAT5 signaling pathway.

Cucurbitacin B and I inhibits colon cancer growth by targeting the Notch signaling pathway.

Cancer stem cells (CSCs) have the ability to self-renew and induce drug resistance and recurrence in colorectal cancer (CRC). As current chemotherapy doesn't eliminate CSCs completely, there is a need to identify novel agents to target them. We investigated the effects of cucurbitacin B (C-B) or I (C-I), a natural compound that exists in edible plants (bitter melons, cucumbers, pumpkins and zucchini), against CRC. C-B or C-I inhibited proliferation, clonogenicity, induced G2/M cell-cycle arrest and caspase-mediated-apoptosis of CRC cells. C-B or C-I suppressed colonosphere formation and inhibited expression of CD44, DCLK1 and LGR5. These compounds inhibited notch signaling by reducing the expression of Notch 1-4 receptors, their ligands (Jagged 1-2, DLL1,3,4), γ-secretase complex proteins (Presenilin 1, Nicastrin), and downstream target Hes-1. Molecular docking showed that C-B or C-I binds to the ankyrin domain of Notch receptor, which was confirmed using the cellular thermal shift assay. Finally, C-B or C-I inhibited tumor xenograft growth in nude mice and decreased the expression of CSC-markers and notch signaling proteins in tumor tissues. Together, our study suggests that C-B and C-I inhibit colon cancer growth by inhibiting Notch signaling pathway.

Diphenylbutylpiperidine Antipsychotic Drugs Inhibit Prolactin Receptor Signaling to Reduce Growth of Pancreatic Ductal Adenocarcinoma in Mice.

BACKGROUND & AIMS: Prolactin (PRL) signaling is up-regulated in hormone-responsive cancers. The PRL receptor (PRLR) is a class I cytokine receptor that signals via the Janus kinase (JAK)-signal transducer and activator of transcription and mitogen-activated protein kinase pathways to regulate cell proliferation, migration, stem cell features, and apoptosis. Patients with pancreatic ductal adenocarcinoma (PDAC) have high plasma levels of PRL. We investigated whether PRLR signaling contributes to the growth of pancreatic tumors in mice. METHODS: We used immunohistochemical analyses to compare levels of PRL and PRLR in multitumor tissue microarrays. We used structure-based virtual screening and fragment-based drug discovery to identify compounds likely to bind PRLR and interfere with its signaling. Human pancreatic cell lines (AsPC-1, BxPC-3, Panc-1, and MiaPaCa-2), with or without knockdown of PRLR (clustered regularly interspaced short palindromic repeats or small hairpin RNA), were incubated with PRL or penfluridol and analyzed in proliferation and spheroid formation. C57BL/6 mice were given injections of UNKC-6141 cells, with or without knockdown of PRLR, into pancreas, and tumor development was monitored for 4 weeks, with some mice receiving penfluridol treatment for 21 days. Human pancreatic tumor tissues were implanted into interscapular fat pads of NSG mice, and mice were given injections of penfluridol daily for 28 days. Nude mice were given injections of Panc-1 cells, xenograft tumors were grown for 2 weeks, and mice were then given intraperitoneal penfluridol for 35 days. Tumors were collected from mice and analyzed by histology, immunohistochemistry, and immunoblots. RESULTS: Levels of PRLR were increased in PDAC compared with nontumor pancreatic tissues. Incubation of pancreatic cell lines with PRL activated signaling via JAK2-signal transducer and activator of transcription 3 and extracellular signal-regulated kinase, as well as formation of pancospheres and cell migration; these activities were not observed in cells with PRLR knockdown. Pancreatic cancer cells with PRLR knockdown formed significantly smaller tumors in mice. We identified several diphenylbutylpiperidine-class antipsychotic drugs as agents that decreased PRL-induced JAK2 signaling; incubation of pancreatic cancer cells with these compounds reduced their proliferation and formation of panco spheres. Injections of 1 of these compounds, penfluridol, slowed the growth of xenograft tumors in the different mouse models, reducing proliferation and inducing autophagy of the tumor cells. CONCLUSIONS: Levels of PRLR are increased in PDAC, and exposure to PRL increases proliferation and migration of pancreatic cancer cells. Antipsychotic drugs, such as penfluridol, block PRL signaling in pancreatic cancer cells to reduce their proliferation, induce autophagy, and slow the growth of xenograft tumors in mice. These drugs might be tested in patients with PDAC.

Targeting transcription factor TCF4 by γ-Mangostin, a natural xanthone.

Given that colon cancer is the third most common cancer in incidence and cause of death in the United States, and current treatment modalities are insufficient, there is a need to develop novel agents. Towards this, here we focus on γ-Mangostin, a bioactive compound present in the Mangosteen (Garcinia mangostana) fruit. γ-Mangostin suppressed proliferation and colony formation, and induced cell cycle arrest and apoptosis of colon cancer cell lines. Further, γ-Mangostin inhibited colonosphere formation. Molecular docking and CETSA (Cellular thermal shift assay) binding assays demonstrated that γ-Mangostin interacts with transcription factor TCF4 (T-Cell Factor 4) at the ß-catenin binding domain with the binding energy of -5.5 Kcal/mol. Moreover, γ-Mangostin treatment decreased TCF4 expression and reduced TCF reporter activity. The compound also suppressed the expression of Wnt signaling target proteins cyclin D1 and c-Myc, and stem cell markers such as LGR5, DCLK1 and CD44. To determine the effect of γ-Mangostin on tumor growth in vivo, we administered nude mice harboring HCT116 tumor xenografts with 5 mg/Kg of γ-Mangostin intraperitoneally for 21 days. γ-Mangostin treatment significantly suppressed tumor growth, with notably lowered tumor volume and weight. In addition, western blot analysis revealed a significant decrease in the expression of TCF4 and its downstream targets such as cyclin D1 and c-Myc. Together, these data suggest that γ-Mangostin inhibits colon cancer growth through targeting TCF4. γ-Mangostin may be a potential therapeutic agent for colon cancer.

Preclinical Pharmacokinetics of Fosciclopirox, a Novel Treatment of Urothelial Cancers, in Rats and Dogs.

Pharmacokinetic studies in rats and dogs were performed to characterize the in vivo performance of a novel prodrug, fosciclopirox. Ciclopirox olamine (CPX-O) is a marketed topical antifungal agent with demonstrated in vitro and in vivo preclinical anticancer activity in several solid tumor and hematologic malignancies. The oral route of administration for CPX-O is not feasible due to low bioavailability and dose-limiting gastrointestinal toxicities. To enable parenteral administration, the phosphoryl-oxymethyl ester of ciclopirox (CPX), fosciclopirox (CPX-POM), was synthesized and formulated as an injectable drug product. In rats and dogs, intravenous CPX-POM is rapidly and completely metabolized to its active metabolite, CPX. The bioavailability of the active metabolite is complete following CPX-POM administration. CPX and its inactive metabolite, ciclopirox glucuronide (CPX-G), are excreted in urine, resulting in delivery of drug to the entire urinary tract. The absolute bioavailability of CPX following subcutaneous administration of CPX-POM is excellent in rats and dogs, demonstrating the feasibility of this route of administration. These studies confirmed the oral bioavailability of CPX-O is quite low in rats and dogs compared with intravenous CPX-POM. Given its broad-spectrum anticancer activity in several solid tumor and hematologic cancers and renal elimination, CPX-POM is being developed for the treatment of urothelial cancer. The safety, dose tolerance, pharmacokinetics, and pharmacodynamics of intravenous CPX-POM are currently being characterized in a United States multicenter first-in-human Phase 1 clinical trial in patients with advanced solid tumors (NCT03348514).

Targeted inhibition of histone deacetylase leads to suppression of Ewing sarcoma tumor growth through an unappreciated EWS-FLI1/HDAC3/HSP90 signaling axis.

Ewing sarcoma (ES) are aggressive pediatric bone and soft tissue tumors driven by EWS-ETS fusion oncogenes, most commonly EWS-FLI1. Treatment of ES patients consists of up to 9 months of alternating courses of 2 chemotherapeutic regimens. Furthermore, EWS-ETS-targeted therapies have yet to demonstrate clinical benefit, thereby emphasizing a clinical responsibility to search for new therapeutic approaches. Our previous in silico drug screening identified entinostat as a drug hit that was predicted to reverse the ES disease signatures and EWS-FLI1-mediated gene signatures. Here, we establish preclinical proof of principle by investigating the in vitro and in vivo efficacy of entinostat in preclinical ES models, as well as characterizing the mechanisms of action and in vivo pharmacokinetics of entinostat. ES cells are preferentially sensitive to entinostat in an EWS-FLI1 or EWS-ERG-dependent manner. Entinostat induces apoptosis of ES cells through G0/G1 cell cycle arrest, intracellular reactive oxygen species (ROS) elevation, DNA damage, homologous recombination (HR) repair impairment, and caspase activation. Mechanistically, we demonstrate for the first time that HDAC3 is a transcriptional target of EWS-FLI1 and that entinostat inhibits growth of ES cells through suppressing a previously unexplored EWS-FLI1/HDAC3/HSP90 signaling axis. Importantly, entinostat significantly reduces tumor burden by 97.4% (89.5 vs. 3397.3 mm3 of vehicle, p < 0.001) and prolongs the median survival of mice (15.5 vs. 8.5 days of vehicle, p < 0.001), in two independent ES xenograft mouse models, respectively. Overall, our studies demonstrate promising activity of entinostat against ES, and support the clinical development of the entinostat-based therapies for children and young adults with metastatic/relapsed ES. KEY MESSAGES: • Entinostat potently inhibits ES both in vitro and in vivo. • EWS-FLI1 and EWS-ERG confer sensitivity to entinostat treatment. • Entinostat suppresses the EWS-FLI1/HDAC3/HSP90 signaling. • HDAC3 is a transcriptional target of EWS-FLI1. • HDAC3 is essential for ES cell viability and genomic stability maintenance.

MIF family proteins in genitourinary cancer: tumorigenic roles and therapeutic potential.

Genitourinary cancers encompass some of the most common solid tumours and have high rates of morbidity and mortality. Inflammation is associated with enhanced tumorigenesis, and a number of pro-inflammatory mediators, such as macrophage migration inhibitory factor (MIF), also promote tumorigenesis. Studies of the role of MIF (which largely functions via the type II transmembrane receptor CD74) in prostate, bladder and kidney cancers suggest that it is a pro-tumorigenic factor in genitourinary malignancy. Inhibiting MIF activity in cell culture and in preclinical animal models of genitourinary cancers reduces the phenotypic hallmarks of cancer, such as proliferation, angiogenesis and tumour aggressiveness, by downregulating signalling pathways such as those regulated by extracellular signal-regulated kinase (ERK), protein kinase B and p53, and MIF may also reverse immunosuppression. Progress has been made in our understanding of the role of MIF (and its family member D-dopachrome tautomerase (DDT)) in genitourinary cancers and how it can be therapeutically targeted.

Metastatic Tumor-in-a-Dish, a Novel Multicellular Organoid to Study Lung Colonization and Predict Therapeutic Response.

Metastasis is a major cause of cancer-related deaths. A dearth of preclinical models that recapitulate the metastatic microenvironment has impeded the development of therapeutic agents that are effective against metastatic disease. Because the majority of solid tumors metastasize to the lung, we developed a multicellular lung organoid that mimics the lung microenvironment with air sac-like structures and production of lung surfactant protein. We used these cultures, called primitive lung-in-a-dish (PLiD), to recreate metastatic disease using primary and established cancer cells. The metastatic tumor-in-a-dish (mTiD) cultures resemble the architecture of metastatic tumors in the lung, including angiogenesis. Pretreating PLiD with tumor exosomes enhanced cancer cell colonization. We next tested the response of primary and established cancer cells to current chemotherapeutic agents and an anti-VEGF antibody in mTiD against cancer cells in two-dimensional (2D) or 3D cultures. The response of primary patient-derived colon and ovarian tumor cells to therapy in mTiD cultures matched the response of the patient in the clinic, but not in 2D or single-cell-type 3D cultures. The sensitive mTiD cultures also produced significantly lower circulating markers for cancer similar to that seen in patients who responded to therapy. Thus, we have developed a novel method for lung colonization in vitro, a final stage in tumor metastasis. Moreover, the technique has significant utility in precision/personalized medicine, wherein this phenotypic screen can be coupled with current DNA pharmacogenetics to identify the ideal therapeutic agent, thereby increasing the probability of response to treatment while reducing unnecessary side effects. SIGNIFICANCE: A lung organoid that exhibits characteristics of a normal human lung is developed to study the biology of metastatic disease and therapeutic intervention.

Comparative oncology approach to drug repurposing in osteosarcoma.

BACKGROUND: Osteosarcoma is an orphan disease for which little improvement in survival has been made since the late 1980s. New drug discovery for orphan diseases is limited by the cost and time it takes to develop new drugs. Repurposing already approved FDA-drugs can help overcome this limitation. Another limitation of cancer drug discovery is the lack of preclinical models that accurately recapitulate what occurs in humans. For OS using dogs as a model can minimize this limitation as OS in canines develops spontaneously, is locally invasive and metastasizes to the lungs as it does in humans. METHODS: In our present work we used high-throughput screens to identify drugs from a library of 2,286 FDA-approved drugs that demonstrated selective growth inhibition against both human and canine OS cell lines. The identified lead compound was then tested for synergy with 7 other drugs that have demonstrated activity against OS. These results were confirmed with in vitro assays and an in vivo murine model of OS. RESULTS: We identified 13 drugs that demonstrated selective growth inhibition against both human and canine OS cell lines. Auranofin was selected for further in vitro combination drug screens. Auranofin showed synergistic effects with vorinostat and rapamycin on OS viability and apoptosis induction. Auranofin demonstrated single-agent growth inhibition in both human and canine OS xenografts, and cooperative growth inhibition was observed in combination with rapamycin or vorinostat. There was a significant decrease in Ki67-positive cells and an increase in cleaved caspase-3 levels in tumor tissues treated with a combination of auranofin and vorinostat or rapamycin. CONCLUSIONS: Auranofin, alone or in combination with rapamycin or vorinostat, may be useful new treatment strategies for OS. Future studies may evaluate the efficacy of auranofin in dogs with OS as a prelude to human clinical evaluation.

Cooperative p16 and p21 action protects female astrocytes from transformation.

Mechanisms underlying sex differences in cancer incidence are not defined but likely involve dimorphism (s) in tumor suppressor function at the cellular and organismal levels. As an example, sexual dimorphism in retinoblastoma protein (Rb) activity was shown to block transformation of female, but not male, murine astrocytes in which neurofibromin and p53 function was abrogated (GBM astrocytes). Correlated sex differences in gene expression in the murine GBM astrocytes were found to be highly concordant with sex differences in gene expression in male and female GBM patients, including in the expression of components of the Rb and p53 pathways. To define the basis of this phenomenon, we examined the functions of the cyclin dependent kinase (CDK) inhibitors, p16, p21 and p27 in murine GBM astrocytes under conditions that promote Rb-dependent growth arrest. We found that upon serum deprivation or etoposide-induced DNA damage, female, but not male GBM astrocytes, respond with increased p16 and p21 activity, and cell cycle arrest. In contrast, male GBM astrocytes continue to proliferate, accumulate chromosomal aberrations, exhibit enhanced clonogenic cell activity and in vivo tumorigenesis; all manifestations of broad sex differences in cell cycle regulation and DNA repair. Differences in tumorigenesis disappeared when female GBM astrocytes are also rendered null for p16 and p21. These data elucidate mechanisms underlying sex differences in cancer incidence and demonstrate sex-specific effects of cytotoxic and targeted therapeutics. This has critical implications for lab and clinical research.

Pharmacokinetics of two 6-mercaptopurine liquid formulations in children with acute lymphoblastic leukemia.

BACKGROUND: A liquid formulation of 6-mercaptopurine (6-MP) was recently approved by the Food and Drug Administration (Purixan®) based on bioavailability (BA) data from healthy adults. We examined the pharmacokinetics (PK) and BA of 6-MP in children with acute lymphoblastic leukemia (ALL) comparing a marketed tablet, two extemporaneously prepared liquid formulations, and data from the approved liquid formulation. METHODS: Twenty-two children (6-17 years) participated in a randomized two-way, crossover study of two cohorts. Group 1 (n = 11; five males) received a 5 mg/ml liquid formulation and the marketed 50 mg 6-MP tablet on separate occasions, and Group 2 (n = 11; five males) received a 50 mg/ml liquid formulation and the marketed tablet. The usual prescribed 6-MP dose (25-115 mg/m2 ) was given after an 8-hr fast. Serial blood samples were collected over 8 hr postdose. Plasma 6-MP concentrations were determined using a good laboratory practice (GLP)-validated liquid chromatography-tandem mass spectrometry method. PK parameters were calculated using noncompartmental analysis and compared within and between cohorts, and thiopurine methyltransferase (TPMT) genotype was analyzed. RESULTS: No patient had a TPMT genotype reflective of a poor metabolizer phenotype. Comparison of PK parameters between 5 and 50 mg/ml treatments revealed significant differences (P <0.05) in AUCN (where AUC is area under the curve), CmaxN , and Tmax . Comparisons within each group revealed significant differences in AUC0-∞ and Tmax in the 5 mg/ml group. CONCLUSIONS: Pharmacokinetic profiles of 6-MP established in healthy adults with the approved liquid formulation may not reflect the PK profile in children with ALL. Formulation-specific differences in PK may significantly impact the dose-exposure profile in these children and must be considered.