Racial differences in RAD51 expression are regulated by miRNA-214-5P and its inhibition synergizes with olaparib in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) affects young women and is the most aggressive subtype of breast cancer (BC). TNBCs disproportionally affect women of African-American (AA) descent compared to other ethnicities. We have identified DNA repair gene RAD51 as a poor prognosis marker in TNBC and its posttranscriptional regulation through microRNAs (miRNAs). This study aims to delineate the mechanisms leading to RAD51 upregulation and develop novel therapeutic combinations to effectively treat TNBCs and reduce disparity in clinical outcomes. METHODS: Analysis of TCGA data for BC cohorts using the UALCAN portal and PrognoScan identified the overexpression of RAD51 in TNBCs. miRNA sequencing identified significant downregulation of RAD51-targeting miRNAs miR-214-5P and miR-142-3P. RT-PCR assays were used to validate the levels of miRNAs and RAD51, and immunohistochemical and immunoblotting techniques were used similarly for RAD51 protein levels in TNBC tissues and cell lines. Luciferase assays were performed under the control of RAD51 3'-UTR to confirm that miR-214-5P regulates RAD51 expression. To examine the effect of miR-214-5P-mediated downregulation of RAD51 on homologous recombination (HR) in TNBC cells, Dr-GFP reporter assays were performed. To assess the levels of olaparib-induced DNA damage responses in miR-214-5P, transfected cells, immunoblots, and immunofluorescence assays were used. Furthermore, COMET assays were used to measure DNA lesions and colony assays were performed to assess the sensitivity of BRCA-proficient TNBC cells to olaparib. RESULTS: In-silico analysis identified upregulation of RAD51 as a poor prognostic marker in TNBCs. miRNA-seq data showed significant downregulation of miR-214-5P and miR-142-3P in TNBC cell lines derived from AA women compared to Caucasian-American (CA) women. miR-214-5P mimics downregulated RAD51 expression and induces HR deficiency as measured by Dr-GFP assays in these cell lines. Based on these results, we designed a combination treatment of miR-214-5P and olaparib in HR-proficient AA TNBC cell lines using clonogenic survival assays. The combination of miR-214-5P and olaparib showed synergistic lethality compared to individual treatments in these cell lines. CONCLUSIONS: Our studies identified a novel epigenetic regulation of RAD51 in TNBCs by miR-214-5P suggesting a novel combination therapies involving miR-214-5P and olaparib to treat HR-proficient TNBCs and to reduce racial disparity in therapeutic outcomes.

Identification of androgen response-related lncRNAs in prostate cancer.

BACKGROUND: Long noncoding RNAs (lncRNAs) are RNA molecules with over 200 nucleotides that do not code for proteins, but are known to be widely expressed and have key roles in gene regulation and cellular functions. They are also found to be involved in the onset and development of various cancers, including prostate cancer (PCa). Since PCa are commonly driven by androgen regulated signaling, mainly stimulated pathways, identification and determining the influence of lncRNAs in androgen response is useful and necessary. LncRNAs regulated by the androgen receptor (AR) can serve as potential biomarkers for PCa. In the present study, gene expression data analysis were performed to distinguish lncRNAs related to the androgen response pathway. METHODS AND RESULTS: We used publicly available RNA-sequencing and ChIP-seq data to identify lncRNAs that are associated with the androgen response pathway. Using Universal Correlation Coefficient (UCC) and Pearson Correlation Coefficient (PCC) analyses, we found 15 lncRNAs that have (a) highly correlated expression with androgen response genes in PCa and are (b) differentially expressed in the setting of treatment with an androgen agonist as well as antagonist compared to controls. Using publicly available ChIP-seq data, we investigated the role of androgen/AR axis in regulating expression of these lncRNAs. We observed AR binding in the promoter regions of 5 lncRNAs (MIR99AHG, DUBR, DRAIC, PVT1, and COLCA1), showing the direct influence of AR on their expression and highlighting their association with the androgen response pathway. CONCLUSION: By utilizing publicly available multiomics data and by employing in silico methods, we identified five candidate lncRNAs that are involved in the androgen response pathway. These lncRNAs should be investigated as potential biomarkers for PCa.

Serine-threonine Kinase Receptor-Associated Protein is a Critical Mediator of APC Mutation-Induced Intestinal Tumorigenesis Through a Feed-Forward Mechanism.

BACKGROUND & AIMS: Inactivation of the Apc gene is a critical early event in the development of sporadic colorectal cancer (CRC). Expression of serine-threonine kinase receptor-associated protein (STRAP) is elevated in CRCs and is associated with poor outcomes. We investigated the role of STRAP in Apc mutation-induced intestinal tumor initiation and progression. METHODS: We generated Strap intestinal epithelial knockout mice (StrapΔIEC) by crossing mice containing floxed alleles of Strap (Strapfl/fl) with Villin-Cre mice. Then we generated ApcMin/+;Strapfl/fl;Vill-Cre (ApcMin/+;StrapΔIEC) mice for RNA-sequencing analyses to determine the mechanism of function of STRAP. We used human colon cancer cell lines (DLD1, SW480, and HT29) and human and mouse colon tumor-derived organoids for STRAP knockdown and knockout and overexpression experiments. RESULTS: Strap deficiency extended the average survival of ApcMin/+ mice by 80 days and decreased the formation of intestinal adenomas. Expression profiling revealed that the intestinal stem cell signature, the Wnt/ß-catenin signaling, and the MEK/ERK pathway are down-regulated in Strap-deficient adenomas and intestinal organoids. Correlation studies suggest that these STRAP-associated oncogenic signatures are conserved across murine and human colon cancer. STRAP associates with MEK1/2, promotes binding between MEK1/2 and ERK1/2, and subsequently induces the phosphorylation of ERK1/2. STRAP activated Wnt/ß-catenin signaling through MEK/ERK-induced phosphorylation of LRP6. STRAP was identified as a target of mutated Apc and Wnt/ß-catenin signaling as chromatin immunoprecipitation and luciferase assays revealed putative binding sites of the ß-catenin/TCF4 complex on the Strap promoter. CONCLUSIONS: STRAP is a target of, and is required in, Apc mutation/deletion-induced intestinal tumorigenesis through a novel feed-forward STRAP/MEK-ERK/Wnt-ß-catenin/STRAP regulatory axis.

Developing 3D Organoid Raft Cultures from Patient-Derived Xenografts as Rapid Models to Screen Efficacy of Experimental Therapeutics.

Reliable preclinical models are needed for screening new cancer drugs. Thus, we developed an improved 3D tumor organoid model termed "organoid raft cultures" (ORCs). Development of ORCs involved culturing tumors ex vivo on collagen beds (boats) with grid supports to maintain their morphological structure. The ORCs were developed from patient-derived xenografts (PDXs) of colon cancers excised from immune-deficient mice (NOD/SCID/IL2Rgammanull). We utilized these new models to evaluate the efficacy of an investigational drug, Navitoclax (ABT-263). We tested the efficacy of ABT-263, an inhibitor of BCL-2 family proteins, in these ORCs derived from a PDX that showed high expression of antiapoptotic BCL2 family proteins (BCL-2, BCL-XL, and BCL-W). Hematoxylin and eosin staining evaluation of PDXs and corresponding ORCs indicated the retention of morphological and other histological integrity of ORCs. ORCs treated with ABT-263 showed decreased expression of antiapoptotic proteins (BCL2, BCL-XL and BCL-W) and increased proapoptotic proteins (BAX and PUMA), with concomitant activation of caspase 3. These studies support the usefulness of the ORCs, developed from PDXs, as an alternative to PDXs and as faster screening models.

Inclusiveness and ethical considerations for observational, translational, and clinical cancer health disparity research.

BACKGROUND: Although general trends in cancer outcomes are improving, racial/ethnic disparities in patient outcomes continue to widen, suggesting disparity-related shortcomings in cancer research designs. METHODS: Using convenience sampling, a total of 24 data sources, representing several research designs and 5 high-burden tumor types, were included for analyses. The percentages of races/ethnicities across each design/tumor type were compared with those of the 2017 US Census data. The authors used a framework based on the Belmont principles to evaluate the ethical strengths and/or weaknesses of each design. RESULTS: In all designs, white individuals were found to be overrepresented. African American and Asian individuals were underrepresented, and Native Americans had consistently poor or no representation. In general, ethical concerns varied according to the study design. Clinical trials were high with regard to respect for persons and beneficence but low for equitable subject selection related to the inclusion of race/ethnicity. Observational study designs were more inclusive for race/ethnicity compared with clinical and translational studies, but their clinical usefulness was less. CONCLUSIONS: The authors proposed that ethical concerns should vary according to the study design. Because observational designs have strengths in inclusiveness for race/ethnicity, their clinical usefulness can be improved by extending the Learning Health System in hospital catchment populations, the use of health records linked to biospecimens, and minority oversampling. Likewise, minority enrollment into clinical trials can be improved through Learning Health System linking and by using National Cancer Institute-mandated Community Outreach and Engagement Cores. This will allow precision medicine for otherwise overlooked minority subgroups.

Wnt receptor Frizzled 8 is a target of ERG in prostate cancer.

Prostate cancer (PCa) is one of the most frequently diagnosed cancers among men. Many molecular changes have been detailed during PCa progression. The gene encoding the transcription factor ERG shows recurrent rearrangement, resulting in the overexpression of ERG in the majority of prostate cancers. Overexpression of ERG plays a critical role in prostate oncogenesis and development of metastatic disease. Among the downstream effectors of ERG, Frizzled family member FZD4 has been shown to be a target of ERG. Frizzled-8 (FZD8) has been shown to be involved in PCa bone metastasis. In the present study, we show that the expression of FZD8 is directly correlated with ERG expression in PCa. Furthermore, we show that ERG directly targets and activates FZD8 by binding to its promoter. This activation is specific to ETS transcription factor ERG and not ETV1. We propose that ERG overexpression in PCa leads to induction of Frizzled family member FZD8, which is known to activate the Wnt pathway. Taken together, these findings uncover a novel mechanism for PCa metastasis, and indicate that FZD8 may represent a potential therapeutic target for PCa.

Single molecule mtDNA fiber FISH for analyzing numtogenesis.

Somatic human cells contain thousands of copies of mitochondrial DNA (mtDNA). In eukaryotes, natural transfer of mtDNA into the nucleus generates nuclear mitochondrial DNA (NUMT) copies. We name this phenomenon as "numtogenesis". Numtogenesis is a well-established evolutionary process reported in various sequenced eukaryotic genomes. We have established a molecular tool to rapidly detect and analyze NUMT insertions in whole genomes. To date, NUMT analyses depend on deep genome sequencing combined with comprehensive computational analyses of the whole genome. This is time consuming, cumbersome and cost prohibitive. Further, most laboratories cannot accomplish such analyses due to limited skills. We report the development of single-molecule mtFIBER FISH (fluorescence in situ hybridization) to study numtogenesis. The development of mtFIBER FISH should aid in establishing a role for numtogenesis in cancers and other human diseases. This novel technique should help distinguish and monitor cancer stages and progression, aid in elucidation of basic mechanisms underlying tumorigenesis and facilitate analyses of processes related to early detection of cancer, screening and/or cancer risk assessment.

Bromoethylindole (BEI-9) redirects NF-κB signaling induced by camptothecin and TNFα to promote cell death in colon cancer cells.

Chemotherapeutic regimens containing camptothecin (CPT), 5-fluorouracil, and oxaliplatin are used to treat advanced colorectal cancer. We previously reported that an indole derivative, 3-(2-bromoethyl)indole (BEI-9), inhibited the proliferation of colon cancer cells and suppressed NF-κB activation. Here, we show that a combination of BEI-9 with either CPT or tumor necrosis factor alpha (TNFα) enhances cell death. Using colorectal cancer cells, we examined the activation of NF-κB by drugs, the potential of BEI-9 for inhibiting drug-induced NF-κB activation, and the enhancement of cell death by combination treatments. Cells were treated with the chemotherapeutic drugs alone or in combination with BEI-9. NF-κB activation, cell cycle profiles, DNA-damage response, markers of cell death signaling and targets of NF-κB were evaluated to determine the effects of single and co-treatments. The combination of BEI-9 with CPT or TNFα inhibited NF-κB activation and reduced the expression of NF-κB-responsive genes, Bcl-xL and COX2. Compared to CPT or BEI-9 alone, sequential treatment of the cells with CPT and BEI-9 significantly enhanced caspase activation and cell death. Co-treatment with TNFα and BEI-9 also caused more cytotoxicity than TNFα or BEI-9 alone. Combined BEI-9 and TNFα enhanced cell death through caspase activation and cleavage of the switch-protein, RIP1 kinase. BEI-9 reduced the expression of COX2 both alone and in combination with CPT or TNF. We postulate that BEI-9 enhances the effects of these drugs on cancer cells by turning off or redirecting NF-κB signaling. Therefore, the combination of BEI-9 with drugs that activate NF-κB needs to be evaluated for clinical applications.

Prolonged exposure of resveratrol induces reactive superoxide species-independent apoptosis in murine prostate cells.

Nitric oxide, a signaling molecule, inhibits mitochondrial respiration by binding with cytochrome c oxidase, resulting in elevated production of reactive superoxide species (reactive oxygen and nitrogen) in the mitochondria and increased susceptibility to cell death. Generation of mitochondrial superoxide species can be suppressed by natural compounds such as resveratrol, a dietary polyphenol found in the skin of red fruits. In various cancer cells, resveratrol shows anti-oxidant and cancer preventive properties. Since, the effect of resveratrol on reactive superoxide species-independent apoptosis in prostate cancer cells is not well illustrated; therefore, we investigated this phenomenon in TRAMP murine prostate cancer cells. To accomplish this, TRAMP cells were incubated with resveratrol, resveratrol + DETA-NONOate, DETA-NONOate (nitric oxide donor), resveratrol + L-NMMA, or L-NMMA (nitric oxide inhibitor) for 48 h, and reactive superoxide species in the mitochondria and culture supernatant were measured. In addition, the mitochondrial membrane potential, cell viability, expression of apoptotic markers (Bax and Bcl2), γ-H2A.x, p53, and caspase-3 was determined. We found that resveratrol suppressed reactive superoxide species such as reactive oxygen species in the mitochondria and nitric oxide in culture supernatant when compared to the DETA-NONOate treatment and disrupted the mitochondrial membrane potential. Resveratrol also reduced cell viability, altered the expression of apoptotic markers (Bax and Bcl2), and increased expression of γ-H2A.x (indicative marker of DNA fragmentation) and p53 (a critical DNA damage response protein). However, there was no appreciable modulation of the caspase-3. Therefore, our data suggest that resveratrol induces superoxide species-independent apoptosis and may act as a therapeutic agent against prostate cancer.

Label-free detection of DNA hybridization with a compact LSPR-based fiber-optic sensor.

A miniaturized, robust, localized surface plasmon resonance (LSPR)-coupled fiber-optic (FO) nanoprobe providing an integrated and portable solution for detection of DNA hybridization and measurement of DNA concentrations has been demonstrated. The FO nanoprobe was created by constructing arrays of metallic nanostructures on the end facets of optical fibers utilizing nanofabrication technologies, including electron beam lithography and lift-off processes. The LSPR-FO nanoprobe device offers real-time, label-free, and low-sample-volume quantification of single-strand DNA in water with high sensitivity and selectivity, achieving a limit of detection around 10 fM. These results demonstrate the feasibility of the LSPR-FO nanoprobe device as a compact and low-cost biosensor for detection of short-strand DNA.

A comparison of 12-gene colon cancer assay gene expression in African American and Caucasian patients with stage II colon cancer.

BACKGROUND: African American (AA) colon cancer patients have a worse prognosis than Caucasian (CA) colon cancer patients, however, reasons for this disparity are not well understood. To determine if tumor biology might contribute to differential prognosis, we measured recurrence risk and gene expression using the Oncotype DX® Colon Cancer Assay (12-gene assay) and compared the Recurrence Score results and gene expression profiles between AA patients and CA patients with stage II colon cancer. METHODS: We retrieved demographic, clinical, and archived tumor tissues from stage II colon cancer patients at four institutions. The 12-gene assay and mismatch repair (MMR) status were performed by Genomic Health (Redwood City, California). Student's t-test and the Wilcoxon rank sum test were used to compare Recurrence Score data and gene expression data from AA and CA patients (SAS Enterprise Guide 5.1). RESULTS: Samples from 122 AA and 122 CA patients were analyzed. There were 118 women (63 AA, 55 CA) and 126 men (59 AA, 67 CA). Median age was 66 years for AA patients and 68 for CA patients. Age, gender, year of surgery, pathologic T-stage, tumor location, the number of lymph nodes examined, lymphovascular invasion, and MMR status were not significantly different between groups (p = 0.93). The mean Recurrence Score result for AA patients (27.9 ± 12.8) and CA patients (28.1 ± 11.8) was not significantly different and the proportions of patients with high Recurrence Score values (≥41) were similar between the groups (17/122 AA; 15/122 CA). None of the gene expression variables, either single genes or gene groups (cell cycle group, stromal group, BGN1, FAP, INHBA1, Ki67, MYBL2, cMYC and GADD45B), was significantly different between the racial groups. After controlling for clinical and pathologic covariates, the means and distributions of Recurrence Score results and gene expression profiles showed no statistically significant difference between patient groups. CONCLUSION: The distribution of Recurrence Score results and gene expression data was similar in a cohort of AA and CA patients with stage II colon cancer and similar clinical characteristics, suggesting that tumor biology, as represented by the 12-gene assay, did not differ between patient groups.

Nanomagnetic levitation three-dimensional cultures of breast and colorectal cancers.

BACKGROUND: Innovative technologies for drug discovery and development, cancer models, stem cell research, tissue engineering, and drug testing in various cell-based platforms require an application similar to the in vivo system. MATERIALS AND METHODS: We developed for the first time nanomagnetically levitated three-dimensional (3-D) cultures of breast cancer (BC) and colorectal cancer (CRC) cells using carbon-encapsulated cobalt magnetic nanoparticles. BC and CRC xenografts grown in severe combined immunodeficient (SCID) mice were evaluated for N-cadherin and epidermal growth factor receptor expressions. These phenotypes were compared with two-dimensional and 3-D cultures grown in a gel matrix. RESULTS: The BC and CRC cells grown by magnetic levitation formed microtissues. The levitated cultures had high viability and were maintained in culture for long periods of time. It has been observed that N-cadherin and epidermal growth factor receptor activities were highly expressed in the levitated 3-D tumor spheres and xenografts of CRC and BC cells. CONCLUSIONS: Nanomagnetically levitated 3-D cultures tend to form stable microtissues of BC and CRC and maybe more feasible for a range of applications in drug discovery or regenerative medicine.

IND-2, a pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline derivative, circumvents multi-drug resistance and causes apoptosis in colon cancer cells.

Naturally occurring condensed quinolines have anticancer properties. In efforts to find active analogues, we designed and synthesized eight polycyclic heterocycles with a pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline framework (IND series). The compounds were evaluated for activity against colon (HCT-116 and S1-MI-80), prostate (PC3 and DU-145), breast (MCF-7 and MDAMB-231), ovarian (ov2008 and A2780), and hepatocellular (HepG2) cancer cells and against non-cancerous Madin Darby canine kidney (MDCK), mouse embryonic fibroblast (NIH/3T3), and human embryonic kidney cells (HEK293). IND-2, a 4-chloro-2-methyl pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline, exhibited more than ten-fold selectivity and potent cytotoxic activity against colon cancer cells relative to the other cancer and non-cancer cells. With five additional colon cancer cell lines (HT-29, HCT-15, LS-180, LS-174, and LoVo), IND-2 had similar cytotoxicity and selectivity, and sub-micromolar concentrations caused changes in the morphology of HCT-116 and HCT-15 cells. IND-2 did not activate the transactivating function of the pregnane X receptor (PXR), indicating that it does not induce PXR-regulated ABCB1 or ABCG2 transporters. Indeed, IND-2 was not a substrate of ABCB1 or ABCG2, and it induced cytotoxicity in HEK293 cells overexpressing ABCB1 or ABCG2 to the same extent as in normal HEK293 cells. IND-2 was cytotoxic to resistant colon carcinoma S1-MI-80 cells, approximately three- and five-fold more than SN-38 and topotecan, respectively. In HCT-116 colon cancer cells, IND-2 produced concentration-dependent changes in mitochondrial membrane potential, leading to apoptosis, and sub-micromolar concentrations caused chromosomal DNA fragmentation. These findings suggest that, by increasing apoptosis, IND-2 has potential therapeutic efficacy for colorectal cancer.

Variable NF-κB pathway responses in colon cancer cells treated with chemotherapeutic drugs.

BACKGROUND: The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway is activated in cells exposed to various stimuli, including those originating on the cell surface or in the nucleus. Activated NF-κB signaling is thought to enhance cell survival in response to these stimuli, which include chemotherapy and radiation. In the present effort, we determined which anticancer drugs preferentially activate NF-κB in colon cancer cells. METHODS: NF-κB reporter cells were established and treated with 5-fluorouracil (5-FU, DNA/RNA damaging), oxaliplatin (DNA damaging), camptothecin (CTP, topoisomerase inhibitor), phleomycin (radiomimetic), or erlotinib (EGFR inhibitor). The activation of NF-κB was assessed by immunofluorescence for p65 translocation, luciferase assays, and downstream targets of NF-κB activation (cIAP2, and Bcl-XL) were evaluated by immunoblotting, by ELISA (CXCL8 and IL-6 in culture supernatants), or by gene expression analysis. RESULTS: Colon cancer cells responded variably to different classes of therapeutic agents, and these agents initiated variable responses among different cell types. CPT activated NF-κB in SW480 colon cancer cells in a dose-dependent manner, but not in HCT116 cells that were either wild-type or deficient for p53. In SW480 colon cancer cells, NF-κB activation by CPT was accompanied by secretion of the cytokine CXCL8, but not by up-regulation of the anti-apoptotic genes, cIAP2 or Bcl-XL. On the contrary, treatment of HCT116 cells with CPT resulted in up-regulation of CXCR2, a receptor for CXCL8, without an increase in cytokine levels. In SW480 cells, NF-κB reporter activity, but not cytokine secretion, was inhibited by SM-7368, an NF-κB inhibitor. CONCLUSION: The results show that, in response to cancer therapeutic agents, NF-κB activation varies with the cellular make up and that drug-induced NF-κB activation may be functionally uncoupled from anti-apoptotic outcomes found for other stimuli. Some cancer cells in a heterogeneous tumor tissue may, under therapeutic pressure, release soluble factors that have paracrine activity on neighboring cells that express the cognate receptors.

Unlocking the Potential of Therapy-Induced Cytokine Responses: Illuminating New Pathways in Cancer Precision Medicine.

Precision cancer medicine primarily aims to identify individual patient genomic variations and exploit vulnerabilities in cancer cells to select suitable patients for specific drugs. These genomic features are commonly determined by gene sequencing prior to therapy, to identify individuals who would be most responsive. This precision approach in cancer therapeutics remains a powerful tool that benefits a smaller pool of patients, sparing others from unnecessary treatments. A limitation of this approach is that proteins, not genes, are the ultimate effectors of biological functions, and therefore the targets of therapeutics. An additional dimension in precision medicine that considers an individual's cytokine response to cancer therapeutics is proposed. Cytokine responses to therapy are multifactorial and vary among individuals. Thus, precision is dictated by the nature and magnitude of cytokine responses in the tumor microenvironment exposed to therapy. This review highlights cytokine responses as modules for precision medicine in cancer therapy, including potential challenges. For solid tumors, both detectability of cytokines in tissue fluids and their being amenable to routine sensitive analyses could address the difficulty of specimen collection for diagnosis and monitoring. Therefore, in precision cancer medicine, cytokines offer rational targets that can be utilized to enhance the efficacy of cancer therapy.

Targeting of oncogenic AAA-ATPase TRIP13 reduces progression of pancreatic ductal adenocarcinoma.

Thyroid hormone receptor-interacting protein 13 (TRIP13) is involved in cancer progression, but its role in pancreatic ductal adenocarcinoma (PDAC) is unknown. Thus, we assessed the expression, functional role, and mechanism of action of TRIP13 in PDAC. We further examined the efficacy of TRIP13 inhibitor, DCZ0415, alone or in combination with gemcitabine on malignant phenotypes, tumor progression, and immune response. We found that TRIP13 was overexpressed in human PDACs relative to corresponding normal pancreatic tissues. TRIP13 knockdown or treatment of PDAC cells with DCZ0415 reduced proliferation and colony formation, and induced G2/M cell cycle arrest and apoptosis. Additionally, TRIP13 knockdown or targeting with DCZ0415 reduced the migration and invasion of PDAC cells by increasing E-cadherin and decreasing N-cadherin and vimentin. Pharmacologic targeting or silencing of TRIP13 also resulted in reduce expression of FGFR4 and STAT3 phosphorylation, and downregulation of the Wnt/ß-catenin pathway. In immunocompromised mouse models of PDAC, knockdown of TRIP13 or treatment with DCZ0415 reduced tumor growth and metastasis. In an immunocompetent syngeneic PDAC model, DCZ0415 treatment enhanced the immune response by lowering expression of PD1/PDL1, increasing granzyme B/perforin expression, and facilitating infiltration of CD3/CD4 T-cells. Further, DCZ0415 potentiated the anti-metastatic and anti-tumorigenic activities of gemcitabine by reducing proliferation and angiogenesis and by inducing apoptosis and the immune response. These preclinical findings show that TRIP13 is involved in PDAC progression and targeting of TRIP13 augments the anticancer effect of gemcitabine.

Combination of dual JAK/HDAC inhibitor with regorafenib synergistically reduces tumor growth, metastasis, and regorafenib-induced toxicity in colorectal cancer.

BACKGROUND: Treatment with regorafenib, a multiple-kinase inhibitor, to manage metastatic colorectal cancers (mCRCs) shows a modest improvement in overall survival but is associated with severe toxicities. Thus, to reduce regorafenib-induced toxicity, we used regorafenib at low concentration along with a dual JAK/HDAC small-molecule inhibitor (JAK/HDACi) to leverage the advantages of both JAK and HDAC inhibition to enhance antitumor activity. The therapeutic efficacy and safety of the combination treatment was evaluated with CRC models. METHODS: The cytotoxicity of JAK/HDACi, regorafenib, and their combination were tested with normal colonic and CRC cells exhibiting various genetic backgrounds. Kinomic, ATAC-seq, RNA-seq, cell cycle, and apoptosis analyses were performed to evaluate the cellular functions/molecular alterations affected by the combination. Efficacy of the combination was assessed using patient-derived xenograft (PDX) and experimental metastasis models of CRC. To evaluate the interplay between tumor, its microenvironment, and modulation of immune response, MC38 syngeneic mice were utilized. RESULTS: The combination therapy decreased cell viability; phosphorylation of JAKs, STAT3, EGFR, and other key kinases; and inhibited deacetylation of histone H3K9, H4K8, and alpha tubulin proteins. It induced cell cycle arrest at G0-G1 phase and apoptosis of CRC cells. Whole transcriptomic analysis showed that combination treatment modulated molecules involved in apoptosis, extracellular matrix-receptor interaction, and focal adhesion pathways. It synergistically reduces PDX tumor growth and experimental metastasis, and, in a syngeneic mouse model, the treatment enhances the antitumor immune response as evidenced by higher infiltration of CD45 and cytotoxic cells. Pharmacokinetic studies showed that combination increased the bioavailability of regorafenib. CONCLUSIONS: The combination treatment was more effective than with regorafenib or JAK/HDACi alone, and had minimal toxicity. A clinical trial to evaluate this combination for treatment of mCRCs is warranted.

Differential Responses to Immune Checkpoint Inhibitors are Governed by Diverse Mismatch Repair Gene Alterations.

PURPOSE: The response to immune checkpoint inhibitors (ICI) in deficient mismatch repair (dMMR) colorectal cancer and endometrial cancer is variable. Here, we explored the differential response to ICIs according to different mismatch repair alterations. EXPERIMENTAL DESIGN: Colorectal cancer (N = 13,701) and endometrial cancer (N = 3,315) specimens were tested at Caris Life Sciences. Median overall survival (mOS) was estimated using Kaplan-Meier. The prediction of high-, intermediate-, and low-affinity epitopes by tumor mutation burden (TMB) values was conducted using R-squared (R2). RESULTS: Compared with mutL (MLH1 and PMS2) co-loss, the mOS was longer in mutS (MSH2 and MSH6) co-loss in all colorectal cancer (54.6 vs. 36 months; P = 0.0.025) and endometrial cancer (81.5 vs. 48.2 months; P < 0.001) patients. In ICI-treated patients, the mOS was longer in mutS co-loss in colorectal cancer [not reached (NR) vs. 36 months; P = 0.011). In endometrial cancer, the mOS was NR vs. 42.2 months; P = 0.711]. The neoantigen load (NAL) in mutS co-loss compared with mutL co-loss was higher in colorectal cancer (high-affinity epitopes: 25.5 vs. 19; q = 0.017, intermediate: 39 vs. 32; q = 0.004, low: 87.5 vs. 73; q < 0.001) and endometrial cancer (high-affinity epitopes: 15 vs. 11; q = 0.002, intermediate: 27.5 vs. 19; q < 0.001, low: 59 vs. 41; q < 0.001), respectively. R2 ranged from 0.25 in mutS co-loss colorectal cancer to 0.95 in mutL co-loss endometrial cancer. CONCLUSIONS: Patients with mutS co-loss experienced longer mOS in colorectal cancer and endometrial cancer and better response to ICIs in colorectal cancer. Among all explored biomarkers, NAL was higher in mutS co-loss and may be a potential driving factor for the observed better outcomes. TMB did not reliably predict NAL.

A Novel Pan-RAS Inhibitor with a Unique Mechanism of Action Blocks Tumor Growth in Mouse Models of GI Cancer.

Here, we describe a novel pan-RAS inhibitor, ADT-007, that potently inhibited the growth of RAS mutant cancer cells irrespective of the RAS mutation or isozyme. RAS WT cancer cells with activated RAS from upstream mutations were equally sensitive. Conversely, cells from normal tissues or RAS WT cancer cells harboring downstream BRAF mutations were insensitive. Insensitivity to ADT-007 was attributed to low activated RAS levels and metabolic deactivation by UDP-glucuronosyltransferases expressed in normal cells but repressed in RAS mutant cancer cells. Cellular, biochemical, and biophysical experiments show ADT-007 binds nucleotide-free RAS to block GTP activation of RAS and MAPK/AKT signaling. Local administration of ADT-007 strongly inhibited tumor growth in syngeneic immune-competent and xenogeneic immune-deficient mouse models of colorectal and pancreatic cancer while activating innate and adaptive immunity in the tumor immune microenvironment. Oral administration of ADT-007 prodrug inhibited tumor growth, supporting further development of this novel class of pan-RAS inhibitors for treating RAS-driven cancers. SIGNIFICANCE: ADT-007 is a 1 st -in-class pan-RAS inhibitor with ultra-high potency and unique selectivity for cancer cells with mutant or activated RAS capable of circumventing resistance and activating antitumor immunity. Further development of ADT-007 analogs or prodrugs with oral bioavailability as a generalizable monotherapy or combined with immunotherapy is warranted.

Molecular Subtyping and Survival Analysis of Osteosarcoma Reveals Prognostic Biomarkers and Key Canonical Pathways.

Osteosarcoma (OS) is a common bone malignancy in children and adolescents. Although histological subtyping followed by improved OS treatment regimens have helped achieve favorable outcomes, a lack of understanding of the molecular subtypes remains a challenge to characterize its genetic heterogeneity and subsequently to identify diagnostic and prognostic biomarkers for developing effective treatments. In the present study, global analysis of DNA methylation, and mRNA and miRNA gene expression in OS patient samples were correlated with their clinical characteristics. The mucin family of genes, MUC6, MUC12, and MUC4, were found to be highly mutated in the OS patients. Results revealed the enrichment of molecular pathways including Wnt signaling, Calcium signaling, and PI3K-Akt signaling in the OS tumors. Survival analyses showed that the expression levels of several genes such as RAMP1, CRIP1, CORT, CHST13, and DDX60L, miRNAs and lncRNAs were associated with survival of OS patients. Molecular subtyping using Cluster-Of-Clusters Analysis (COCA) for mRNA, lncRNA, and miRNA expression; DNA methylation; and mutation data from the TARGET dataset revealed two distinct molecular subtypes, each with a distinctive gene expression profile. Between the two subtypes, three upregulated genes, POP4, HEY1, CERKL, and seven downregulated genes, CEACAM1, ABLIM1, LTBP2, ISLR, LRRC32, PTPRF, and GPX3, associated with OS metastasis were found to be differentially regulated. Thus, the molecular subtyping results provide a strong basis for classification of OS patients that could be used to develop better prognostic treatment strategies.