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Lingfan Xu, Enze Ma, Tao Zeng, Ruya Zhao, Yulei Tao, Xufeng Chen, Jeff Groth, Chaozhao Liang, Hailiang Hu and Jiaoti Huang

ATM is a well-known master regulator of double strand break (DSB) DNA repair and the defective DNA repair has been therapeutically exploited to develop PARP inhibitors based on the synthetic lethality strategy. ATM mutation is found with increased prevalence in advanced metastatic castration-resistant prostate cancer (mCRPC). However, the molecular mechanisms underlying ATM mutation-driving disease progression are still largely unknown. Here, we report that ATM mutation contributes to the CRPC progression through a metabolic rather than DNA repair mechanism. We showed that ATM deficiency generated by CRISPR/Cas9 editing promoted CRPC cell proliferation and xenograft tumor growth. ATM deficiency altered cellular metabolism and enhanced Warburg effect in CRPC cells. We demonstrated that ATM deficiency shunted the glucose flux to aerobic glycolysis by upregulating LDHA expression, which generated more lactate and produced less mitochondrial ROS to promote CRPC cell growth. Inhibition of LDHA by siRNA or inhibitor FX11 generated less lactate and accumulated more ROS in ATM-deficient CRPC cells and therefore potentiated the cell death of ATM-deficient CRPC cells. These findings suggest a new therapeutic strategy for ATM-mutant CRPC patients by targeting LDHA-mediated glycolysis metabolism, which might be effective for the PARP inhibitor resistant mCRPC tumors.

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Han-Wei Lin, Ying-Cheng Chiang, Nai-Yun Sun, Yu-Li Chen, Chi-Fang Chang, Yi-Jou Tai, Chi-An Chen and Wen-Fang Cheng

The role of chitinase-3-like protein 1 (CHI3L1) in ovarian cancer and the possible mechanisms were elucidated. CHI3L1 is a secreted glycoprotein and associated with inflammation, fibrosis, asthma, extracellular tissue remodeling and solid tumors. Our previous study showed CHI3L1 could be a potential prognostic biomarker for epithelial ovarian cancer and could protect cancer cells from apoptosis. Therefore, clinical data and quantitation of CHI3L1 of ovarian cancer patients, tumor spheroid formation, side-population assays, Aldefluor and apoptotic assays, ELISA, RT-PCR, immunoblotting and animal experiments were performed in two ovarian cancer cells lines, OVCAR3 and CA5171, and their CHI3L1-overexpressing and -knockdown transfectants. High expression of CHI3L1 was associated with poor outcome and chemoresistance in ovarian cancer patients. The mRNA expression of CHI3L1 in CA5171 ovarian cancer stem-like cells was 3-fold higher than in CA5171 parental cells. CHI3L1 promoted the properties of ovarian cancer stem-like cells including generating more and larger tumor spheroids and a higher percentage of ALDH+ in tumor cells and promoting resistance to cytotoxic drug-induced apoptosis. CHI3L1 could induce both the Akt (essential) and Erk signaling pathways, and then enhance expression of β-catenin followed by SOX2, and finally promote tumor spheroid formation and other properties of ovarian cancer stem-like cells. OVCAR3 CHI3L1-overexpressing transfectants were more tumorigenic in vivo, whereas CA5171 CHI3L1-knockdown transfectants were not tumorigenic in vivo. CHI3L1 critically enhances the properties of ovarian cancer stem-like cells. CHI3L1 or CHI3L1-regulated signaling pathways and molecules could be potential therapeutic targets in ovarian cancer.

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Veronica Vella, Maria Luisa Nicolosi, Patrizia Cantafio, Michele Massimino, Rosamaria Lappano, Paolo Vigneri, Roberto Ciuni, Pietro Gangemi, Andrea Morrione, Roberta Malaguarnera and Antonino Belfiore

Patients with thyroid cancers refractory to radioiodine (RAI) treatment show a limited response to various therapeutic options and a low survival rate. The recent use of multikinase inhibitors has also met limited success. An alternative approach relies on drugs that induce cell differentiation, as the ensuing increased expression of the cotransporter for sodium and iodine (NIS) may partially restore sensitivity to radioiodine. The inhibition of the ERK1/2 pathway has shown some efficacy in this context. Aggressive thyroid tumors overexpress the isoform-A of the insulin receptor (IR-A) and its ligand IGF-2; this IGF-2/IR-A loop is associated with de-differentiation and stem-like phenotype, resembling RAI-refractory tumors. Importantly, IR-A has been shown to be positively modulated by the non-integrin collagen receptor DDR1 in human breast cancer. Using undifferentiated human thyroid cancer cells, we now evaluated the effects of DDR1 on IGF-2/IR-A loop and on markers of cell differentiation and stemness. DDR1 silencing or downregulation caused significant reduction of IR-A and IGF-2 expression, and concomitant increased levels of differentiation markers (NIS, Tg, TSH, TPO). Conversely, markers of epithelial-to-mesenchymal transition (Vimentin, Snail-2, Zeb1, Zeb2 and N-Cadherin) and stemness (OCT-4, SOX-2, ABCG2 and Nanog) decreased. These effects were collagen independent. In contrast, overexpression of either DDR1 or its kinase-inactive variant K618A DDR1-induced changes suggestive of less differentiated and stem-like phenotype. Collagen stimulation was uneffective. In conclusion, in poorly differentiated thyroid cancer, DDR1 silencing or downregulation blocks the IGF-2/IR-A autocrine loop and induces cellular differentiation. These results may open novel therapeutic approaches for thyroid cancer.

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Simon Linder, Henk G van der Poel, Andries M Bergman, Wilbert Zwart and Stefan Prekovic

The androgen receptor drives the growth of metastatic castration-resistant prostate cancer. This has led to the development of multiple novel drugs targeting this hormone-regulated transcription factor, such as enzalutamide – a potent androgen receptor antagonist. Despite the plethora of possible treatment options, the absolute survival benefit of each treatment separately is limited to a few months. Therefore, current research efforts are directed to determine the optimal sequence of therapies, discover novel drugs effective in metastatic castration-resistant prostate cancer and define patient subpopulations that ultimately benefit from these treatments. Molecular studies provide evidence on which pathways mediate treatment resistance and may lead to improved treatment for metastatic castration-resistant prostate cancer. This review provides, firstly a concise overview of the clinical development, use and effectiveness of enzalutamide in the treatment of advanced prostate cancer, secondly it describes translational research addressing enzalutamide response vs resistance and lastly highlights novel potential treatment strategies in the enzalutamide-resistant setting.

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Nicole Panarelli, Kathrin Tyryshkin, Justin Jong Mun Wong, Adrianna Majewski, Xiaojing Yang, Theresa Scognamiglio, Michelle Kang Kim, Kimberly Bogardus, Thomas Tuschl, Yao-Tseng Chen and Neil Renwick

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) can be challenging to evaluate histologically. MicroRNAs (miRNAs) are small RNA molecules that often are excellent biomarkers due to their abundance, cell-type and disease stage specificity and stability. To evaluate miRNAs as adjunct tissue markers for classifying and grading well-differentiated GEP-NETs, we generated and compared miRNA expression profiles from four pathological types of GEP-NETs. Using quantitative barcoded small RNA sequencing and state-of-the-art sequence annotation, we generated comprehensive miRNA expression profiles from archived pancreatic, ileal, appendiceal and rectal NETs. Following data preprocessing, we randomly assigned sample profiles to discovery (80%) and validation (20%) sets prior to data mining using machine-learning techniques. High expression analyses indicated that miR-375 was the most abundant individual miRNA and miRNA cistron in all samples. Leveraging prior knowledge that GEP-NET behavior is influenced by embryonic derivation, we developed a dual-layer hierarchical classifier for differentiating GEP-NET types. In the first layer, our classifier discriminated midgut (ileum, appendix) from non-midgut (rectum, pancreas) NETs based on miR-615 and -92b expression. In the second layer, our classifier discriminated ileal from appendiceal NETs based on miR-125b, -192 and -149 expression, and rectal from pancreatic NETs based on miR-429 and -487b expression. Our classifier achieved overall accuracies of 98.5% and 94.4% in discovery and validation sets, respectively. We also found provisional evidence that low- and intermediate-grade pancreatic NETs can be discriminated based on miR-328 expression. GEP-NETs can be reliably classified and potentially graded using a limited panel of miRNA markers, complementing morphological and immunohistochemistry-based approaches to histologic evaluation.

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Bruno Heidi Nozima, Thais Biude Mendes, Gustavo José da Silva Pereira, Rodrigo Pinheiro Araldi, Edna Sadayo Miazato Iwamura, Soraya Soubhi Smaili, Gianna Maria Griz Carvalheira and Janete Maria Cerutti

We previously proposed that high expression of FAM129A can be used as a thyroid carcinoma biomarker in preoperative diagnostic exams of thyroid nodules. Here, we identify that FAM129A expression is increased under nutrient and growth factor depletion in a normal thyroid cell line (PCCL3), overlapping with increased expression of autophagy-related protein and inhibition of AKT/mTOR/p70S6K. Supplementation of insulin, TSH and serum to the medium was able to reduce the expression of both FAM129A and autophagy-related protein and reestablish the AKT/mTOR/p70S6K axis. To determine the direct role of FAM129A on autophagy, FAM129A was transfected into PCCL3 cells. Its overexpression induced autophagic vesicles formation, evidenced by transmission electron microscopy. Co-expression of FAM129A and mCherry-EGFP-LC3B in PCCL3 showed an increased yellow puncta formation, suggesting that FAM129Ainduces autophagy. To further confirm its role on autophagy, we knockdown FAM129A in two thyroid carcinoma cell lines (TPC1 and FTC-236). Unexpectedly, FAM129A silencing increased autophagic flux, suggesting that FAM129A inhibits autophagy in these models. We next co-transfected PCCL3 cells with FAM129A and RET/PTC1 and tested autophagy in this context. Co-expression of FAM129A and RET/PTC1 oncogene in PCCL3 cells, inhibited RET/PTC1-induced autophagy. Together, our data suggest that, in normal cells FAM129A induces autophagy in order to maintain cell homeostasis and provide substrates under starvation conditions. Instead, in cancer cells, decreased autophagy may help the cells to overcome cell death. FAM129A regulates autophagy in a cell- and/or context-dependent manner. Our data reinforce the concept that autophagy can be used as a strategy for cancer treatment.

Open access

Milena Doroszko, Marcin Chrusciel, Joanna Stelmaszewska, Tomasz Slezak, Slawomir Anisimowicz, Ursula Plöckinger, Marcus Quinkler, Marco Bonomi, Slawomir Wolczynski, Ilpo Huhtaniemi, Jorma Toppari and Nafis A Rahman

Aberrantly expressed G protein-coupled receptors in tumors are considered as potential therapeutic targets. We analyzed the expressions of receptors of gonadotropin-releasing hormone (GNRHR), luteinizing hormone/chorionic gonadotropin (LHCGR) and follicle-stimulating hormone (FSHR) in human adrenocortical carcinomas and assessed their response to GnRH antagonist therapy. We further studied the effects of the GnRH antagonist cetrorelix acetate (CTX) on cultured adrenocortical tumor (ACT) cells (mouse Cα1 and Y-1, and human H295R), and in vivo in transgenic mice (SV40 T-antigen expression under inhibin α promoter) bearing Lhcgr and Gnrhr in ACT. Both models were treated with control (CT), CTX, human chorionic gonadotropin (hCG) or CTX+hCG, and their growth and transcriptional changes were analyzed. In situ hybridization and qPCR analysis of human adrenocortical carcinomas (n = 11–13) showed expression of GNRHR in 54/73%, LHCGR in 77/100% and FSHR in 0%, respectively. CTX treatment in vitro decreased cell viability and proliferation, and increased caspase 3/7 activity in all treated cells. In vivo, CTX and CTX+hCG (but not hCG alone) decreased ACT weights and serum LH and progesterone concentrations. CTX treatment downregulated the tumor markers Lhcgr and Gata4. Upregulated genes included Grb10, Rerg, Nfatc and Gnas, all recently found to be abundantly expressed in healthy adrenal vs ACT. Our data suggest that CTX treatment may improve the therapy of human adrenocortical carcinomas by direct action on GNRHR-positive cancer cells inducing apoptosis and/or reducing gonadotropin release, directing tumor cells towards a healthy adrenal gene expression profile.

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T Vandamme, M Beyens, G Boons, A Schepers, K Kamp, K Biermann, P Pauwels, W W De Herder, L J Hofland, M Peeters, G Van Camp and K Op de Beeck

Mutations in DAXX/ATRX, MEN1 and genes involved in the phosphoinositide-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway have been implicated in pancreatic neuroendocrine neoplasms (pNENs). However, mainly mutations present in the majority of tumor cells have been identified, while proliferation-driving mutations could be present only in small fractions of the tumor. This study aims to identify high- and low-abundance mutations in pNENs using ultra-deep targeted resequencing. Formalin-fixed paraffin-embedded matched tumor-normal tissue of 38 well-differentiated pNENs was sequenced using a HaloPlex targeted resequencing panel. Novel amplicon-based algorithms were used to identify both single nucleotide variants (SNVs) and insertion-deletions (indels) present in >10% of reads (high abundance) and in <10% of reads (low abundance). Found variants were validated by Sanger sequencing. Sequencing resulted in 416,711,794 reads with an average target base coverage of 2663 ± 1476. Across all samples, 32 high-abundance somatic, 3 germline and 30 low-abundance mutations were withheld after filtering and validation. Overall, 92% of high-abundance and 84% of low-abundance mutations were predicted to be protein damaging. Frequently, mutated genes were MEN1, DAXX, ATRX, TSC2, PI3K/Akt/mTOR and MAPK-ERK pathway-related genes. Additionally, recurrent alterations on the same genomic position, so-called hotspot mutations, were found in DAXX, PTCH2 and CYFIP2. This first ultra-deep sequencing study highlighted genetic intra-tumor heterogeneity in pNEN, by the presence of low-abundance mutations. The importance of the ATRX/DAXX pathway was confirmed by the first-ever pNEN-specific protein-damaging hotspot mutation in DAXX. In this study, both novel genes, including the pro-apoptotic CYFIP2 gene and hedgehog signaling PTCH2, and novel pathways, such as the MAPK-ERK pathway, were implicated in pNEN.

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Lautaro Zubeldía-Brenner, Catalina De Winne, Sofía Perrone, Santiago A Rodríguez-Seguí, Christophe Willems, Ana María Ornstein, Isabel Lacau-Mengido, Hugo Vankelecom, Carolina Cristina and Damasia Becu-Villalobos

Preclinical and clinical studies support that Notch signaling may play an important oncogenic role in cancer, but there is scarce information for pituitary tumors. We therefore undertook a functional study to evaluate Notch participation in pituitary adenoma growth. Tumors generated in Nude mice by subcutaneous GH3 somatolactotrope cell injection were treated in vivo with DAPT, a γ-secretase inhibitor, thus inactivating Notch signaling. This treatment led to pituitary tumor reduction, lower prolactin and GH tumor content and a decrease in angiogenesis. Furthermore, in silico transcriptomic and epigenomic analyses uncovered several tumor suppressor genes related to Notch signaling in pituitary tissue, namely Btg2, Nr4a1, Men1, Zfp36 and Cnot1. Gene evaluation suggested that Btg2, Nr4a1 and Cnot1 may be possible players in GH3 xenograft growth. Btg2 mRNA expression was lower in GH3 tumors compared to the parental line, and DAPT increased its expression levels in the tumor in parallel with the inhibition of its volume. Cnot1 mRNA levels were also increased in the pituitary xenografts by DAPT treatment. And the Nr4a1 gene was lower in tumors compared to the parental line, though not modified by DAPT. Finally, because DAPT in vivo may also be acting on tumor microenvironment, we determined the direct effect of DAPT on GH3 cells in vitro. We found that DAPT decreases the proliferative, secretory and migration potential of GH3 cells. These results position selective interruption of Notch signaling as a potential therapeutic tool in adjuvant treatments for aggressive or resistant pituitary tumors.

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Wei Guan, Junhui Hu, Lu Yang, Ping Tan, Zhuang Tang, Brian L West, Gideon Bollag, Hua Xu and Lily Wu

For men with castration-resistant prostate cancer (CRPC), androgen-deprivation therapy (ADT) often becomes ineffective requiring the addition of docetaxel, a proven effective chemotherapy option. Tumor-associated macrophages (TAMs) are known to provide protumorigenic influences that contribute to treatment failure. In this study, we examined the contribution of TAMs to docetaxel treatment. An increased infiltration of macrophages in CRPC tumors was observed after treatment with docetaxel. Prostate cancer cells treated with docetaxel released more macrophage colony-stimulating factor (M-CSF-1 or CSF-1), IL-10 and other factors, which can recruit and modulate circulating monocytes to promote their protumorigenic functions. Inhibition of CSF-1 receptor kinase signaling with a small molecule antagonist (PLX3397) in CRPC models significantly reduces the infiltration of TAMs and their influences. As such, the addition of PLX3397 to docetaxel treatment resulted in a more durable tumor growth suppression than docetaxel alone. This study reveals a rational strategy to abrogate the influences of TAMs and extend the treatment response to docetaxel in CRPC.