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Cetuximab is the sole anti-EGFR monoclonal antibody that is FDA approved to treat head and neck squamous cell carcinoma (HNSCC). However, no predictive biomarkers of cetuximab response are known for HNSCC. Herein, we address the molecular mechanisms underlying cetuximab resistance in an in vitro model. We established a cetuximab resistant model (FaDu), using increased cetuximab concentrations for more than eight months. The resistance and parental cells were evaluated for cell viability and functional assays. Protein expression was analyzed by Western blot and human cell surface panel by lyoplate. The mutational profile and copy number alterations (CNA) were analyzed using whole-exome sequencing (WES) and the NanoString platform. FaDu resistant clones exhibited at least two-fold higher IC50 compared to the parental cell line. WES showed relevant mutations in several cancer-related genes, and the comparative mRNA expression analysis showed 36 differentially expressed genes associated with EGFR tyrosine kinase inhibitors resistance, RAS, MAPK, and mTOR signaling. Importantly, we observed that overexpression of KRAS, RhoA, and CD44 was associated with cetuximab resistance. Protein analysis revealed EGFR phosphorylation inhibition and mTOR increase in resistant cells. Moreover, the resistant cell line demonstrated an aggressive phenotype with a significant increase in adhesion, the number of colonies, and migration rates. Overall, we identified several molecular alterations in the cetuximab resistant cell line that may constitute novel biomarkers of cetuximab response such as mTOR and RhoA overexpression. These findings indicate new strategies to overcome anti-EGFR resistance in HNSCC., This work was supported by Barretos Cancer Hospital and the Public Ministry of Labor Campinas (Research, Prevention, and Education of Occupational Cancer) in Campinas, Brazil, CAPESDFATD (88887.137283/2017-00). INFG is the recipient of a FAPESP Ph.D. fellowship (2017/22305-9).

Tumor-infiltrating lymphocytes include heterogeneous populations of T lymphocytes that play crucial roles in the tumor immune response; importantly, their presence in the tumor tissue may predict clinical outcomes. Therefore, we herein studied the prognostic significance of the presence and location of CD3+, CD8+, and FoxP3+ T lymphocytes in colorectal cancer samples. In the intratumor analysis, our data did not reveal any association between lymphocyte infiltrations with clinical or pathological data. However, in the tumor margins, we found that the presence of high infiltrations of CD3+, CD8+, or FoxP3+ T lymphocytes were associated with TNM stages I-II (p = 0.021, p = 0.022, and p = 0.012, respectively) and absence of lymph node metastases (p = 0.010, p = 0.003, and p = 0.004, respectively). Despite these associations with good prognostic indicators, we were not able to find any statistically significant alterations in the overall survival of the patients, even though high infiltrations of FoxP3+ T lymphocytes in the tumor margins resulted in an increased overall survival of 14 months. Taken together, these data show that the presence of CD3+, CD8+, or FoxP3+T lymphocyte infiltrates in the tumor margins are associated with the pathogenesis of CRC, but only high Foxp3+ T lymphocyte infiltrations in the tumor invasive margins are inclined to indicate favorable prognosis., This work was supported by National funds through the Foundation for Science and Technology (FCT)—projects PTDC/MED-ONC/28658/2017, UIDB/50026/2020, and UIDP/50026/2020; and by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) projects NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023. AMB was supported by the FCT fellowship SFRH/BD/120371/2016 and ET by the FCT investigator grant IF/01390/2014 and Estímulo Individual ao Emprego Científico CEECIND/03070/2020.

Neuroendocrine neoplasms (NENs) are the most common tumor of the appendix and have an excellent prognosis. Appendiceal tumors diagnosed between 1989 and 2019 were reviewed, and clinical data were collected from patient files. Part of the series was immuno-profiled for markers related to cell cycle proliferation and/or senescence-type, apoptotic, and metastatic potential. Appendix NENs were detected in 74 patients, with 0.47% of incidence per appendectomy. The median age of the patients was 21.5 years, with two age peaks of incidence at 17.0 and 55.2 years. The median tumors size was 5.8 mm, and most were smaller than 10 mm. Lymphovascular and perineural invasion, as well as necrosis, was associated with larger tumor size. G1 tumors composed 96.0% of the cohort. The presence of moderate/strong p16 and the absent/low Bcl-2 expression was frequently observed and associated with a smaller size. This study represents one of the largest cohorts and with a long follow-up. For tumors smaller than 10 mm appendicectomy was sufficient as a curative procedure, as revealed by the good outcome. This series presented a 100% disease-free survival. The indolent phenotype of appendix NENs is supported by the expression of markers that point towards a strong inhibition of cell replication and growth inhibition., This study was supported by FCT—Fundação para a Ciência e a Tecnologia, Ministério da Ciência, Tecnologia e Ensino Superior by a research contract to João Vinagre (CEECIND/00201/2017). Further funding was obtained from the project “Advancing cancer research: from basic knowledge to application” NORTE-01-0145-FEDER-000029: “Projetos Estruturados de I & D & I”, funded by Norte 2020—Programa Operacional Regional do Norte. Additional funding was obtained from the project PTDC/MED-ONC/31438/2017 (The other faces of Telomerase: Looking beyond tumor immortalization), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), COMPETE 2020—Operational Programme for Competitiveness and Internationalization (POCI) and by Portuguese funds through FCT under project POCI-01-0145-FEDER-016390: CANCEL STEM”.

The following are available online at http://www.mdpi.com/2073-4409/9/2/485/s1, Figure S1: Validation of WNK2 overexpression by RT-PCR., Autophagy is a cell-survival pathway with dual role in tumorigenesis, promoting either tumor survival or tumor death. WNK2 gene, a member of the WNK (with no lysine (K)) subfamily, acts as a tumor suppressor gene in gliomas, regulating cell migration and invasion; however, its role in autophagy process is poorly explored. The WNK2-methylated human glioblastoma cell line A172 WT (wild type) was compared to transfected clones A172 EV (empty vector), and A172 WNK2 (WNK2 overexpression) for the evaluation of autophagy using an inhibitor (bafilomycin A1—baf A1) and an inducer (everolimus) of autophagic flux. Western blot and immunofluorescence approaches were used to monitor autophagic markers, LC3A/B and SQSTM1/p62. A172 WNK2 cells presented a significant decrease in LC3B and p62 protein levels, and in LC3A/B ratio when compared with control cells, after treatment with baf A1 + everolimus, suggesting that WNK2 overexpression inhibits the autophagic flux in gliomas. The mTOR pathway was also evaluated under the same conditions, and the observed results suggest that the inhibition of autophagy mediated by WNK2 occurs through a mTOR-independent pathway. In conclusion, the evaluation of the autophagic process demonstrated that WNK2 inhibits the autophagic flux in glioblastoma cell line., This project was supported by the Barretos Cancer Hospital Internal Research Funds (PAIP) to Rui Manuel Reis and by the Public Ministry of Labor Campinas (Research, Prevention, and Education of Occupational Cancer Project), Campinas, Brazil. Ana Laura Vieira Alves is the recipient of a FAPESP master fellowship (2016/18907-0).

RAF kinase inhibitory protein (RKIP) is an important modulator of several signaling pathways, whose loss of expression has been observed in different human malignancies. In several studies, this decreased expression has been associated not only with the presence of metastasis, which leads to RKIP's identification as a metastasis suppressor gene, but also with tumor aggressiveness and poor prognosis. Importantly, RKIP has been reported to be involved in the modulation of cancer therapy response trough different mechanisms that sensitize cells to apoptosis. Given these findings, the usage of compounds capable of regulating RKIP expression emerged as a possible therapeutic approach to modulate therapy response and to control tumor aggressiveness. Herein, we will provide updated information about RKIP expression in different solid tumors and how it affects prognosis and response to therapy. Moreover, concerning therapy response we will present revised information about the compounds reported as being RKIP's modulators and the RKIP-dependent mechanisms, through which some of these compounds modulate drugs response.

The identification of signaling pathways that are involved in gliomagenesis is crucial for targeted therapy design. In this study we assessed the biological and therapeutic effect of ingenol-3-dodecanoate (IngC) on glioma. IngC exhibited dose-time-dependent cytotoxic effects on large panel of glioma cell lines (adult, pediatric cancer cells, and primary cultures), as well as, effectively reduced colonies formation. Nevertheless, it was not been able to attenuate cell migration, invasion, and promote apoptotic effects when administered alone. IngC exposure promoted S-phase arrest associated with p21CIP/WAF1 overexpression and regulated a broad range of signaling effectors related to survival and cell cycle regulation. Moreover, IngC led glioma cells to autophagy by LC3B-II accumulation and exhibited increased cytotoxic sensitivity when combined to a specific autophagic inhibitor, bafilomycin A1. In comparison with temozolomide, IngC showed a mean increase of 106-fold in efficacy, with no synergistic effect when they were both combined. When compared with a known compound of the same class, namely ingenol-3-angelate (I3A, Picato®), IngC showed a mean 9.46-fold higher efficacy. Furthermore, IngC acted as a potent inhibitor of protein kinase C (PKC) activity, an emerging therapeutic target in glioma cells, showing differential actions against various PKC isotypes. These findings identify IngC as a promising lead compound for the development of new cancer therapy and they may guide the search for additional PKC inhibitors., This research received was funded by Amazonia Fitomedicamentos Ltda, and Barretos Cancer Hospital, all from Brazil.

Glioblastoma (GBM) is the most frequent and aggressive type of brain tumor. There are limited therapeutic options for GBM so that new and effective agents are urgently needed. Euphol is a tetracyclic triterpene alcohol, and it is the main constituent of the sap of the medicinal plant Euphorbia tirucalli. We previously identified anti-cancer activity in euphol based on the cytotoxicity screening of 73 human cancer cells. We now expand the toxicological screening of the inhibitory effect and bioactivity of euphol using two additional glioma primary cultures. Euphol exposure showed similar cytotoxicity against primary glioma cultures compared to commercial glioma cells. Euphol has concentration-dependent cytotoxic effects on cancer cell lines, with more than a five-fold difference in the IC50 values in some cell lines. Euphol treatment had a higher selective cytotoxicity index (0.64-3.36) than temozolomide (0.11-1.13) and reduced both proliferation and cell motility. However, no effect was found on cell cycle distribution, invasion and colony formation. Importantly, the expression of the autophagy-associated protein LC3-II and acidic vesicular organelle formation were markedly increased, with Bafilomycin A1 potentiating cytotoxicity. Finally, euphol also exhibited antitumoral and antiangiogenic activity in vivo, using the chicken chorioallantoic membrane assay, with synergistic temozolomide interactions in most cell lines. In conclusion, euphol exerted in vitro and in vivo cytotoxicity against glioma cells, through several cancer pathways, including the activation of autophagy-associated cell death. These findings provide experimental support for further development of euphol as a novel therapeutic agent for GBM, either alone or in combination chemotherapy., The work was supported by the Amazonia Fitomedicamentos (FITO05/2012) Ltda. and Barretos Cancer Hospital, all from Brazil.

Anti-VEGF therapy with Bevacizumab is approved for glioblastoma treatment, however, it is known that tumors acquired resistance and eventually became even more aggressive and infiltrative after treatment. In the present study we aimed to unravel the potential cellular mechanisms of resistance to Bevacizumab in glioblastoma in vitro models. Using a panel of glioblastoma cell lines we found that Bevacizumab is able to block the secreted VEGF by the tumor cells and be internalized to the cytoplasm, inducing cytotoxicity in vitro. We further found that Bevacizumab increases the expression of hypoxic (HIF-1α and CAIX) and glycolytic markers (GLUT1 and MCT1), leading to higher glucose uptake and lactate production. Furthermore, we showed that part of the consumed glucose by the tumor cells can be stored as glycogen, hampering cell dead following Bevacizumab treatment. Importantly, we found that this change on the glycolytic metabolism occurs independently of hypoxia and before mitochondrial impairment or autophagy induction. Finally, the combination of Bevacizumab with glucose uptake inhibitors decreased in vivo tumor growth and angiogenesis and shift the expression of glycolytic proteins. In conclusion, we reported that Bevacizumab is able to increase the glucose metabolism on cancer cells by abrogating autocrine VEGF in vitro. Define the effects of anti-angiogenic drugs at the cellular level can allow us to discover ways to revert acquired resistance to this therapeutic approaches in the future, This study was partially developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER), and through the Competitiveness Factors Operational Programme (COMPETE), by Portuguese funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038, and by Brazilian MCTI/CNPq No 73/2013. VMG was recipient from a PhD fellowship (SFRH/BD/51997/2012) from Fundação para a Ciência e Tecnologia (FCT), Portugal. FC was recipient of a master FAPESP fellowship (n° 2014/03684-0) and “BEPE - Bolsa Estágio de Pesquisa no Exterior” (n° 2015/02691-6). OM is recipient of a post-doc fellowship (SFRH/BPD/108351/2015) from FCT, Portugal, info:eu-repo/semantics/publishedVersion

Magnetoliposomes containing calcium ferrite (CaFe2O4) nanoparticles were developed and characterized for the first time. CaFe2O4 nanoparticles were covered by a lipid bilayer or entrapped in liposomes forming, respectively, solid or aqueous magnetoliposomes as nanocarriers for new antitumor drugs. The magnetic nanoparticles were characterized by UV/Visible absorption, XRD, HR-TEM, and SQUID, exhibiting sizes of 5.2 ± 1.2 nm (from TEM) and a superparamagnetic behavior. The magnetoliposomes were characterized by DLS and TEM. The incorporation of two new potential antitumor drugs (thienopyridine derivatives) specifically active against breast cancer in these nanosystems was investigated by fluorescence emission and anisotropy. Aqueous magnetoliposomes, with hydrodynamic diameters around 130 nm, and solid magnetoliposomes with sizes of ca. 170 nm, interact with biomembranes by fusion and are able to transport the antitumor drugs with generally high encapsulation efficiencies (70%). These fully biocompatible drug-loaded magnetoliposomes can be promising as therapeutic agents in future applications of combined breast cancer therapy., This research was funded by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding of CF-UM-UP (UID/FIS/04650/2013; UID/FIS/04650/2019), CQUM (UID/QUI/00686/2016; UID/QUI/00686/2019) and LA-26 (PEst-C/SAU/LA0026/2013), and through the research project PTDC/QUI-QFI/28020/2017 (POCI-01-0145-FEDER-028020), financed by FCT, European Fund of Regional Development (FEDER), COMPETE2020 and Portugal2020. The magnetic measurements were supported by projects UTAP-EXPL/NTec/0046/2017, NORTE-01-0145-FEDER-028538 e PTDC/FIS-MAC/29454/2017. The APC was also funded by FCT. B.D.C. acknowledges FCT for a PhD grant (SFRH/BD/141936/2018).

Cervical cancer is the third most commonly diagnosed tumor type and the fourth cause of cancer-related death in females. Therapeutic options for cervical cancer patients remain very limited. Annona crassiflora Mart. is used in traditional medicine as antimicrobial and antineoplastic agent. However, little is known about its antitumoral properties. In this study the antineoplastic effect of crude extract and derived partitions from A. crassiflora Mart in cervical cancer cell lines was evaluated. The crude extract significantly alters cell viability of cervical cancer cell lines as well as proliferation and migration, and induces cell death in SiHa cells. Yet, the combination of the crude extract with cisplatin leads to antagonistic effect. Importantly, the hexane partition derived from the crude extract presented cytotoxic effect both in vitro and in vivo, and initiates cell responses, such as DNA damage (H2AX activity), apoptosis via intrinsic pathway (cleavage of caspase-9, caspase-3, poly (ADP-ribose) polymerase (PARP) and mitochondrial membrane depolarization) and decreased p21 expression by ubiquitin proteasome pathway. Concluding, this work shows that hexane partition triggers several biological responses such as DNA damage and apoptosis, by intrinsic pathways, and was also able to promote a direct decrease in tumor perimeter in vivo providing a basis for further investigation on its antineoplastic activity on cervical cancer., This study was supported by grants from the FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/2013 -FPXII-BIOPLAT), CAPES, FAPEMIG, UFSJ and Barretos Cancer Hospital, all from Brazil., info:eu-repo/semantics/acceptedVersion

The latex from Euphorbia tirucalli is used in Brazil as a folk medicine for several diseases, including cancer. Recently, we showed a cytotoxic activity of E. tirucalli euphol in a wide range of cancer cell lines. Moreover, we showed that euphol inhibits proliferation, motility and colony formation in pancreatic cancer cells, induces autophagy and sensitizes glioblastoma cells to temozolomide cytotoxicity. Herein, we report in vitro activity of three semi-synthetic ingenol compounds derived from E. tirucalli, IngA (ingenol-3-trans-cinnamate), IngB (ingenol-3-hexanoate) and IngC (ingenol-3-dodecanoate), against a large panel of human cancer cell lines. Antineoplastic effects of the three semi-synthetic compounds were assessed using MTS assays on 70 cancer cell lines from a wide array of solid tumors. Additionally, their antitumor potential was compared with known compounds of the same class, namely ingenol-3-angelate (Picato®) and ingenol 3,20-dibenzoate and in combination with standard chemotherapeutic agents. We observed that IngA, B, and C exhibited dose-dependent cytotoxic effects. Amongst the semi-synthetic compounds, IngC displayed the best activity across the tumor cell lines. In comparison with ingenol-3-angelate and ingenol 3,20-dibenzoate, IngC showed a mean of 6.6 and 3.6-fold higher efficacy, respectively, against esophageal cancer cell lines. Besides, IngC sensitized esophageal cancer cells to paclitaxel treatment. In conclusion, the semi-synthetic ingenol compounds, in particular, IngC, demonstrated a potent antitumor activity on all cancer cell lines evaluated. Although the underlying mechanisms of action of IngC are not elucidated, our results provide insights for further studies suggesting IngC as a putative therapy for cancer treatment., Grants from Amazonia Fitomedicamentos Ltda, and Barretos Cancer Hospital, all from Brazil, supported this study.

Acral lentiginous melanoma (ALM) is the less common subtype with singular characterization. TERT (human telomerase reverse transcriptase) promoter mutations have being described as recurrent in melanomas and infrequent in ALM, but their real incidence and clinical relevance is unclear. The objectives of this study were to describe the prevalence of TERT promoter mutations in ALM, and correlate with the molecular profile of other drive genes and clinical features. Sixty-one samples from 48 patients with ALM were analyzed. After DNA isolation, the mutation profiles of the hotspot region of BRAF, NRAS, KIT, PDGFRA, and TERT genes were determined by PCR amplification followed by direct Sanger sequencing. KIT, PDGFRA, and VEGFR2 gene amplification was performed by quantitative PCR. Clinical information such as survival, clinical stage, and Breslow tumor classification were obtained from medical records. TERT promoter mutations were found in 9.3% of the cases, BRAF in 10.3%, NRAS in 7.5%, KIT in 20.7%, and PDGFRA in 14.8% of ALM. None of the cases showed KIT, PDGFRA, or VEGFR2 gene amplification. We found an association between KIT mutations and advanced Clark level (IV and V, P=0.043) and TERT promoter mutations with low mitotic index. No other significant associations were observed between mutation profile and patients' clinical features nor survival rates. Oncogenic TERT promoter mutations are present in a fraction of ALMs. No relevant associations were found between TERT mutation status and clinical/molecular features nor survival. Mutations of KIT and PDGFRA are the most common genetic alterations, and they can be therapeutic targets for these patients., This project was supported by FAPESP - Brazil (2012/4194-1) to Vinicius de Lima Vazquez., info:eu-repo/semantics/publishedVersion

Objective: Soft tissue sarcomas (STSs) are heterogeneous tumors displaying multiple and complex molecular abnormalities with no specific pattern. Despite current therapeutic advances, the patients with STS still have a poor outcome, which makes it necessary to find out new prognostic markers. The Raf kinase inhibitory protein (RKIP) has been associated with prognosis in several human neoplasms; however, its role in STS is unknown. Methods: In the present study RKIP expression was assessed by immunohistochemistry in a series of 87 STSs, and its expression profile was associated with the patients' pathological parameters. Results: We found that RKIP is expressed in the cytoplasm of the great majority of cases, and absent in only approximately 18% of cases (16/87). Importantly, we observed that loss of RKIP expression was associated with poor outcome, constituting an independent prognostic marker. Conclusion: This is the first study assessing RKIP expression levels in STS. We showed that loss of RKIP expression is present in a small subset of cases; however, its absence was associated with poor survival and may be a potential marker for STS prognosis., This project was partially supported by a CNPq Universal Grant (476192/2013-7) to Rui Manuel Reis. Olga Martinho is re-cipient of a Post-Doc fellowship (SFRH/BPD/108351/2015) from Fundação para a Ciência e Tecnologia (FCT), Portugal.

A large number of classic antineoplastic agents are derived from plants. Euphorbia tirucalli L. (Euphorbiaceae) is a subtropical and tropical plant, used in Brazilian folk medicine against many diseases, including cancer, yet little is known about its true anticancer properties. The present study evaluated the antitumor effect of the tetracyclic triterpene alcohol, euphol, the main constituent of E. tirucalli in a panel of 73 human cancer lines from 15 tumor types. The biological effect of euphol in pancreatic cells was also assessed. The combination index was further used to explore euphol interactions with standard drugs. Euphol showed a cytotoxicity effect against several cancer cell lines (IC50 range, 1.41-38.89 µM), particularly in esophageal squamous cell (11.08 µM) and pancreatic carcinoma cells (6.84 µM), followed by prostate, melanoma, and colon cancer. Cytotoxicity effects were seen in all cancer cell lines, with more than half deemed highly sensitive. Euphol inhibited proliferation, motility and colony formation in pancreatic cancer cells. Importantly, euphol exhibited synergistic interactions with gemcitabine and paclitaxel in pancreatic and esophageal cell lines, respectively. To the best of our knowledge, this study constitutes the largest in vitro screening of euphol efficacy on cancer cell lines and revealed its in vitro anti-cancer properties, particularly in pancreatic and esophageal cell lines, suggesting that euphol, either as a single agent or in combination with conventional chemotherapy, is a potential anti-cancer drug., Amazônia Fitomedicamentos Ltda. (grant no. FITO 05/2012) and Barretos Cancer Hospital, all from Brazil, info:eu-repo/semantics/publishedVersion

Glioblastoma (GBM) is one of the most glycolytic and angiogenic human tumors, characteristics that contribute to the poor prognosis associated with this type of tumor. A lactate shuttle has been described between tumor cells and endothelial cells (ECs), with the monocarboxylate transporters (MCTs) acting as important players in this tumor-EC communication. In this study, we aimed to understand how the tumor microenvironment modulates EC metabolism, and to characterize the role of MCTs in the glioma-brain EC crosstalk. Exposure of human brain microvascular ECs (HBMEC) to GBM cell-conditioned media increased the expression of MCT1, which corresponded to activation of oxidative metabolism and an increase in angiogenic capacity, as determined by increased proliferation, migration, and vessel assembly. Lactate depletion from the microenvironment or inhibition of lactate uptake in HBMEC induced an increase in lactate production and a decrease in proliferation, migration, and vessel assembly. Moreover, addition of lactate to HBMEC media promoted activation of AKT and AMPK pathways and increased expression in NFκB, HIF-1α, and the lactate receptor GPR81. Here, we demonstrate a role for MCT1 as a mediator of lactate signaling between glioma cells and brain ECs. Our results suggest that MCT1 can mediate EC metabolic reprograming, proliferation, and vessel sprouting in response to tumor signaling. Thus, targeting MCT1 in both tumor cells and brain EC may be a promising therapeutic strategy for the treatment of GBM., Foundation for Science and Technology (FCT), Grant number: POCI-01-0145-FEDER007038; MCTI/CNPq, Brazil, Grant number: Nº 73/2013; NORTE 2020, FEDER, COMPETE, Grant number: NORTE-01-0145- FEDER-000013; Fundação para a Ciência e a Tecnologia, Grant number: SFRH/BD/51997/ 2012

Serotonin regulates prostate growth through androgen receptor modulation, Aging and testosterone almost inexorably cause benign prostatic hyperplasia (BPH) in Human males. However, etiology of BPH is largely unknown. Serotonin (5-HT) is produced by neuroendocrine prostatic cells and presents in high concentration in normal prostatic transition zone, but its function in prostate physiology is unknown. Previous evidence demonstrated that neuroendocrine cells and 5-HT are decreased in BPH compared to normal prostate. Here, we show that 5-HT is a strong negative regulator of prostate growth. In vitro, 5-HT inhibits rat prostate branching through down-regulation of androgen receptor (AR). This 5-HT's inhibitory mechanism is also present in human cells of normal prostate and BPH, namely in cell lines expressing AR when treated with testosterone. In both models, 5-HT's inhibitory mechanism was replicated by specific agonists of 5-Htr1a and 5-Htr1b. Since peripheral 5-HT production is specifically regulated by tryptophan hydroxylase 1(Tph1), we showed that Tph1 knockout mice present higher prostate mass and up-regulation of AR when compared to wild-type, whereas 5-HT treatment restored the prostate weight and AR levels. As 5-HT is decreased in BPH, we present here evidence that links 5-HT depletion to BPH etiology through modulation of AR. Serotoninergic prostate pathway should be explored as a new therapeutic target for BPH., Projects NORTE-01-0246-FEDER-000012, NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Program (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) and Bolsa de Investigação GSK Inovação em Urologia 2012, info:eu-repo/semantics/publishedVersion

Zeolites Y (faujasite) and MOR (mordonite) were used as hosts for temozolomide (TMZ), a current good-standard chemotherapeutic agent used in the treatment of glioblastoma brain tumors. TMZ was loaded into zeolites by liquid-phase adsorption at controlled pH. FTIR, 1H NMR, MS, SEM, UV/vis and chemical analysis demonstrated the successful loading of TMZ into zeolite hosts. The hydrolysis of TMZ in MTIC (TMZ metabolite) after the preparation of drug delivery systems (DDS) was observed in simulated body fluid. The effect of zeolites and DDS were evaluated on the viability of glioblastoma cell lines. Unloaded Y zeolite presented toxicity to cancer cells in contrast to MOR. In accordance, the best results in potentiation of the TMZ effect was obtained with MOR. We found that mordonite loaded with 0.026 mmol of TMZ was able to decrease the half maximal inhibitory concentrations (IC50) at least 3-fold in comparison to free temozolomide both in vitro and in vivo., R.A. and N.V. are recipients of PhD fellowships (SFRH/BI/51118/2010 and SFRH/BD/97797/2013) from Fundacao para a Ciencia e a Tecnologia (FCT, Portugal). O.M. is a recipient of a Post-Doc fellowship (UMINHO/BPD/32/2013) from QREN. The authors thank the FCT and FEDER-COMPETE-QREN-EU for financial support to the Research Centers, CQ/UM, PEst-C/QUI/UI0686/2013 (F-COMP-01-0124-FEDER-037302) and Life and Health Sciences Research Institute (ICVS, University of Minho, Portugal). The NMR spectrometer is part of the National NMR Network (RNRMN), supported with funds from FCT/QREN (Quadro de Referencia Estrategico Nacional).

Epidermal growth factor receptor (EGFR) is overexpressed in up to 90% of head and neck squamous cell carcinoma (HNSCC) tumors. Cetuximab is the first targeted (anti-EGFR) therapy approved for the treatment of HNSCC patients. However, its efficacy is limited due to primary and secondary resistance, and there is no predict biomarkers of response. New generation of EGFR inhibitors with pan HER targeting and irreversible action, such as afatinib and allitinib, represents a significant therapeutic promise. In this study, we intend to compare the potential cytotoxicity of two anti-EGFR inhibitors (afatinib and allitinib) with cetuximab and to identify potential predictive biomarkers of response in a panel of HNSCC cell lines. The mutational analysis in the eight HNSCC cell lines revealed an EGFR mutation (p.H773Y) and gene amplification in the HN13 cells. According to the growth inhibition score (GI), allitinib was the most cytotoxic drug, followed by afatinib and finally cetuximab. The higher AKT phosphorylation level was associated with resistance to anti-EGFR agents. Therefore, we further performed drug combinations with anti-AKT agent (MK2206) and AKT1 gene editing, which demonstrated afatinib and allitinib sensitivity restored. Additionally, in silico analysis of TCGA database showed that AKT1 overexpression was present in 14.7% (41/279) of HNSCC cases, and was associated with perineural invasion in advanced stage. In conclusion, allitinib presented a greater cytotoxic profile when compared to afatinib and cetuximab. AKT pathway constitutes a predictive marker of allitinib response and combination with AKT inhibitors could restore response and increase treatment success., FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/2013 - FPXII-BIOPLAT) and the Assistance Program and Incentive Research (PAIP), Barretos Cancer Hospital São Paulo, Brazil. The authors would like to acknowledge the technical support of Gabriela Lamberti in the clonogenic assays. A.L.C and R.M.R are recipients of a National Counsel of Technological and Scientific Development (CNPq) scholarship and O.C.M is recipient of a Portuguese Foundation for Science and Technology (FCT) scholarship (SFRH/BPD/108351/2015), info:eu-repo/semantics/publishedVersion

Background: Glioblastoma, the most frequent and malignant adult brain tumor, has been extensively studied. However, there is no effective treatment, and to overcome this challenging scenario, it is essential to improve preclinical biological models. This study aimed to molecularly characterize short-term glioblastoma primary cultures and to compare them with patient tumor profiles. Methods: Glioblastoma cell lines were established from Barretos Cancer Hospital patients diagnosed with glioblastoma. The cells were cultured with DMEM (+)10% FBS (+)1% PS and were molecularly characterized using array CGH (aCGH), next-generation and Sanger sequencing. Results: We established four short-term glioblastoma cultures and we found that the primary cells exhibited a diversity of chromosomal aberrations, with gain of chromosome 7 and loss of chromosomes 10, 13 and 17p being the most frequent alterations. Mutation profiling showed that hotspot TERT promoter mutations were present in 3/4 cases, followed by mutations in TP53 (2/4) and in the RB1, BRAF and PTEN (1/4) genes. A similar chromosomal and mutation pattern was observed in all short-term cultures and matched frozen tumors. Conclusions: Herein, short-term glioblastoma primary cultures were successfully characterized and had genetic make-ups that were similar to those of patient tumors, suggesting that short-term primary cultures are suitable in vitro models for studies of glioblastoma biology., Universal/CNPq (475358/2011-2-Reis RM), FAPESP (2012/19590-0-Reis RM) and the MCTI/CNPq No. 73/2013 (Reis RM) grants. Bidinotto LT was a recipient of the FAPESP fellowship (2011/08523-7 and 2012/08287-4), info:eu-repo/semantics/publishedVersion

The authors are thankful to Dr. A. S. Azevedo for collecting the powder diffraction data., Two different structures of zeolites, faujasite (FAU) and Linde type A (LTA), were studied to investigate their suitability for drug delivery systems (DDS). The zeolites in the sodium form (NaY and NaA) were used as hosts for encapsulation of α-cyano-4- hydroxycinnamic acid (CHC). CHC, an experimental anticancer drug, was encapsulated in both zeolites by diffusion in liquid phase. These new drug delivery systems, CHC@zeolite, were characterized by spectroscopic techniques (FTIR, 1H NMR, 13C and 27Al solidstate MAS NMR, and UV−vis), chemical analysis, powder X-ray diffraction (XRD) and scanning electron microscopy (SEM). The effect of the zeolites and CHC@zeolite drug deliveries on HCT-15 human colon carcinoma cell line viability was evaluated. Both zeolites alone revealed no toxicity to HCT-15 cancer cells. Importantly, CHC@zeolite exhibit an inhibition of cell viability up to 585-fold, when compared to the non-encapsulated drug. These results indicate the potential of the zeolites for drug loading and delivery into cancer cells to induce cell death, O.M. and R.A. are recipients of fellowships (SFRH/BD/36463/2007, SFRH/BI/51118/2010) from Fundação para a Ciência e a Tecnologia (FCT, Portugal). This work was supported by the FCT projects refs PEst-C/ QUI/UI0686/2011, PEst-C/CTM/LA0011/2011, and PTDC/ SAU-FCF/104347/2008, under the scope of “Programa Operacional Temático Factores de Competitividade” (COMPETE) of “Quadro Comunitário de Apoio III” and cofinanced by Fundo Comunitário Europeu FEDER, and the Centre of Chemistry and Life and Health Sciences Research Institute (University of Minho, Portugal).

Persistent HPV infection alone is not sufficient for cervical cancer development, which requires additional molecular alterations for tumor progression and metastasis ultimately leading to a lethal disease. In this study, we performed a comprehensive analysis of HER family receptor alterations in cervical adenocarcinoma. We detected overexpression of HER protein, mainly HER2, which was an independent prognostic marker for these patients. By using in vitro and in vivo approaches, we provided evidence that HER inhibitors, allitinib and lapatinib, were effective in reducing cervical cancer aggressiveness. Furthermore, combination of these drugs with glucose uptake blockers could overcome the putative HIF1-a-mediated resistance to HER-targeted therapies. Thus, we propose that the use of HER inhibitors in association with glycolysis blockers can be a potentially effective treatment option for HER-positive cervical cancer patients., FINEP (MCTI/FINEP/MS/SCTIE/DECIT-BIOPLAT (1302/13), Brazil and co-funded by the project “ON.2 SR&TD Integrated Program (NORTE-07-0124-FEDER-000017)” co-funded by Programa Operacional Regional do Norte (ON.2- O Novo Norte), Quadro de Referência Estratégico Nacional (QREN), through Fundo Europeu de Desenvolvimento Regional (FEDER). OM is recipient of a post-doc fellowship (SFRH/BPD/108351/2015) from Fundação para a Ciência e Tecnologia (FCT), Portugal. FC is recipient of a master fellowship (2014/03684-0) from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). VMG is recipient from a PhD fellowship (SFRH/BD/51997/2012) from Fundação para a Ciência e Tecnologia (FCT), Portugal, info:eu-repo/semantics/publishedVersion

Epidermal growth factor receptor (EGFR) is overexpressed in up to 90% of head and neck squamous cell carcinoma (HNSCC) tumors. Cetuximab is the first targeted (anti-EGFR) therapy approved for the treatment of HNSCC patients. However, its efficacy is limited due to primary and secondary resistance, and there is no predict biomarkers of response. New generation of EGFR inhibitors with pan HER targeting and irreversible action, such as afatinib and allitinib, represents a significant therapeutic promise. In this study, we intend to compare the potential cytotoxicity of two anti-EGFR inhibitors (afatinib and allitinib) with cetuximab and to identify potential predictive biomarkers of response in a panel of HNSCC cell lines. The mutational analysis in the eight HNSCC cell lines revealed an EGFR mutation (p.H773Y) and gene amplification in the HN13 cells. According to the growth inhibition score (GI), allitinib was the most cytotoxic drug, followed by afatinib and finally cetuximab. The higher AKT phosphorylation level was associated with resistance to anti-EGFR agents. Therefore, we further performed drug combinations with anti-AKT agent (MK2206) and AKT1 gene editing, which demonstrated afatinib and allitinib sensitivity restored. Additionally, in silico analysis of TCGA database showed that AKT1 overexpression was present in 14.7% (41/279) of HNSCC cases, and was associated with perineural invasion in advanced stage. In conclusion, allitinib presented a greater cytotoxic profile when compared to afatinib and cetuximab. AKT pathway constitutes a predictive marker of allitinib response and combination with AKT inhibitors could restore response and increase treatment success.

Background: Glioblastomas (GBM) present a high cellular heterogeneity with conspicuous necrotic regions associated with hypoxia, which is related to tumor aggressiveness. GBM tumors exhibit high glycolytic metabolism with increased lactate production that is extruded to the tumor microenvironment through monocarboxylate transporters (MCTs). While hypoxia-mediated regulation of MCT4 has been characterized, the role of MCT1 is still controversial. Thus, we aimed to understand the role of hypoxia in the regulation of MCT expression and function in GBM, MCT1 in particular. Methods: Expression of hypoxia-and glycolytic-related markers, as well as MCT1 and MCT4 isoforms was assessed in in vitro and in vivo orthotopic glioma models, and also in human GBM tissues by immunofluorescence/immunohistochemistry and Western blot. Following MCT1 inhibition, either pharmacologically with CHC (a-cyano-4-hydroxynnamic acid) or genetically with siRNAs, we assessed GBM cell viability, proliferation, metabolism, migration and invasion, under normoxia and hypoxia conditions. Results: Hypoxia induced an increase in MCT1 plasma membrane expression in glioma cells, both in in vitro and in vivo models. Additionally, treatment with CHC and downregulation of MCT1 in glioma cells decreased lactate production, cell proliferation and invasion under hypoxia. Moreover, in the in vivo orthotopic model and in human GBM tissues, there was extensive co-expression of MCT1, but not MCT4, with the GBM hypoxia marker CAIX. Conclusion: Hypoxia-induced MCT1 supports GBM glycolytic phenotype, being responsible for lactate efflux and an important mediator of cell survival and aggressiveness. Therefore, MCT1 constitutes a promising therapeutic target in GBM., This study was supported by Projecto Estratégico- LA 26 – 2013-2014 (PEst-C/SAL/LA0026/2013) and ON.2 SR&TD Integrated Program (NORTE-07-0124FEDER-000017)” co-funded by Programa Operacional Regional do Norte (ON.2- O Novo Norte), Quadro de Referência Estratégico Nacional (QREN), through Fundo Europeu de Desenvolvimento Regional (FEDER), as well as MCTI/CNPq Nº 73/2013 (Brazil). VMG received a fellowship from Fundação para a Ciência e Tecnologia (FCT) ref. SFRH/BD/51997/2012., info:eu-repo/semantics/publishedVersion

The epidermal growth factor receptor (EGFR) is a member of the HER family of growth factors that activates several intracellular signaling pathways promoting proliferation and survival. EGFR over-expression is frequently associated with gene mutation or amplification, thereby constituting a major target for molecular therapies. Recently, a new generation of EGFR inhibitors has been developed with pan-HER properties and irreversible actions. AllitinibA (R) (AST1306) is an orally active, highly selective irreversible inhibitor of the HER family of receptor tyrosine kinases with promising efficacies. In the present study we aimed to investigate the cytotoxicity of allitinib in a large panel of human cancer-derived cell lines and to correlate its efficacy to the mutational status of the EGFR, KRAS, BRAF, PI3KCA and PTEN genes. In addition, we aimed to evaluate the functional role of KRAS mutations in the response to this new inhibitor. In total 76 different cancer-derived cell lines, representing 11 distinct histological types, were analyzed and classified into three groups: highly sensitive (HS), moderately sensitive (MS) and resistant (R). We found that 28 (36.8 %) cancer-derived cell lines exhibited a HS phenotype, 19 (25.0 %) a MS phenotype and 29 (38.1 %) a R phenotype. Allitinib showed a stronger cytotoxicity in head and neck, esophageal, melanoma and lung cancer-derived cell lines. We found that KRAS mutations were significantly associated with the R phenotype. To substantiate these results, an allitinib-sensitive lung cancer-derived cell line (H292) was transfected with plasmids carrying the two most common activating KRAS mutations (p.G12D and p.G12S). We found that both mutations reverted the allitinib-sensitive phenotype in these cells. The current study represents the largest in vitro assessment of allitinib cytotoxicity performed to date. Through this study, we identified cancer types that could potentially benefit from this drug. Additionally, our findings suggest that prevalent KRAS mutations constitute potential predictive biomarkers for allitinib response., This study was partially supported by FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/2013 - FPXII-BIOPLAT) and the Assistance Program and Incentive Research (PAIP), Barretos Cancer Hospital Sao Paulo, Brazil. A.L.C and R.M.R are recipients of a National Counsel of Technological and Scientific Development (CNPq) scholarship. M.N.R is recipient of a CNPq scholarship (380434/2015-6) and O.C.M is recipient of a Portuguese Foundation for Science and Technology (FCT) scholarship (SFRH/BPD/108351/2015)., info:eu-repo/semantics/publishedVersion

The authors are thankful to Dr. K. Biernacki for DFT calculations and Dr. A.S. Azevedo for collecting the powder diffraction data., The faujasite zeolite structure was studied to investigate its suitability for development of new drug delivery systems (DDS). The sodium form (NaY) of the zeolite was used for encapsulation of a-cyano-4-hydroxy-cinnamic acid (CHC), an experimental anticancer drug used in colorectal cancer therapy. The DDS was prepared by diffusion in liquid phase of CHC as a guest in the void space of the host zeolite structure at pH 7.0. The molecular integrity of CHC in the encapsulation process was evaluated by proton nuclear magnetic resonance spectroscopy(1H NMR) and Ultraviolet–Visible spectroscopy (UV–Vis). The new drug delivery system, CHC@NaY, was characterized by Fourier transform infrared spectroscopy and UV–Vis, chemical analysis, powder X-ray diffraction, and Scanning electron microscopy. Analysis of the data of the drug alone and encapsulated in NaY show that CHC and the zeolite framework preserved their original structure. The effect of the zeolite and DDS on HCT-15 human colon carcinoma cell line viability was evaluated. The encapsulation of CHC significantly increased its potency., OM and RA are recipients of fellowships (SFRH/BD/ 36463/2007, SFRH/BI/51118/2010) from Fundacão para a Ciência e Tecnologia (FCT, Portugal).This study was supported by the Centre of Chemistry and Life and Health Sciences Research Institute (ICVS), University of Minho, Portugal, FCT (Portugal) through POCTI and FEDER projects (ref. POCTI-SFA-3-686) and by the FCT grant ref. PTDC/SAU-FCF/104347/2008, under the scope of ‘‘Programa Operacional Temático "Factores de Competitividade’’ (COMPETE) of ‘‘Quadro Comunitário de Apoio III’’ and co-financed by Fundo Comunitário Europeu FEDER.

Background: Glioblastomas are the most frequent and deadly brain tumors. Genomic alterations, as well as its high heterogeneity, are important features of these tumors. Despite the advances in the biology of these tumors, it is essential to develop models, such as primary tumor cell lines, that better mimic their genomic diversity and therapeutic behavior. We aimed to characterize the molecular profile of two (HCB2 and HCB151) established immortalized glioblastoma cell lines. Methods: Immortalized glioblastoma cell lines, HCB2 and HCB151, were established from Barretos Cancer Hospital patients diagnosed with glioblastoma. The cells were cultured with DMEM+10%BFS+1%PS and molecularly characterized, using arrayCGH, next-generation sequencing (Ion Torrent) and validated by Sanger sequencing, miRNA expression and CNV using the NanoString platform, and BD Lyoplate™ Human Cell Surface Marker Screening Panel using Accuri™ C6 flow cytometer. The tumorigenic capacity was further determined in vivo by the chicken chorioallantoic membrane (CAM) assay, and its response to temozolomide-based chemotherapy was done by MTS. Results: The aCGH analysis showed that the most frequent events was the gain of chromosome 7 and loss of chromosomes 10, 13, and 17p. HCB2 exhibited mutations in the TERT gene, while HCB151 showed mutations in the TP53, PTEN, LZTR1, and TERT genes. The immortalized cultures present an unique miRNA profile. The CAM assay showed that HCB2 and HCB151 were able to form vascularized tumors, with HCB151 more aggressive than HCB2. In addition, both HCB2 (IC50>1000 µM) and HCB151 (IC50=783 µM) showed resistance to TMZ when compared to commercial glioblastomas cell lines (23-441 µM). Conclusion: The present findings showed that the immortalized glioblastoma cell lines showed classic genomic features of glioblastomas and exhibited a similar biologic behavior, suggesting that these cell lines are a good model for glioblastoma preclinical studies. Citation Format: Adriana Cruvinel-Carloni, Renato José da Silva-Oliveira, Viviane Aline Oliveira-Silva, Marcela Nunes Rosa, Lucas Tadeu Bidinotto, Gustavo Noriz Berdinarelli, Raul Torrieri, Adriane Feijó Evangelista, Olga Catarina Martinho, Vera Mónica Gonçalves, Carlos Clara, Gisele Caravina de Almeida, Rui Manuel Reis. Omics profile of two immortalized Brazilian glioblastoma cell cultures [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A29.

Expression, mutation and copy number analysis of platelet-derived growth factor receptor A (PDGFRA) and its ligand PDGFA in gliomas, BACKGROUND: Malignant gliomas are the most prevalent type of primary brain tumours but the therapeutic armamentarium for these tumours is limited. Platelet-derived growth factor (PDGF) signalling has been shown to be a key regulator of glioma development. Clinical trials evaluating the efficacy of anti-PDGFRA therapies on gliomas are ongoing. In this study, we intended to analyse the expression of PDGFA and its receptor PDGFRA, as well as the underlying genetic (mutations and amplification) mechanisms driving their expression in a large series of human gliomas. METHODS: PDGFA and PDGFRA expression was evaluated by immunohistochemistry in a series of 160 gliomas of distinct World Health Organization (WHO) malignancy grade. PDGFRA-activating gene mutations (exons 12, 18 and 23) were assessed in a subset of 86 cases by PCR-single-strand conformational polymorphism (PCR-SSCP), followed by direct sequencing. PDGFRA gene amplification analysis was performed in 57 cases by quantitative real-time PCR (QPCR) and further validated in a subset of cases by chromogenic in situ hybridisation (CISH) and microarray-based comparative genomic hybridisation (aCGH). RESULTS: PDGFA and PDGFRA expression was found in 81.2% (130 out of 160) and 29.6% (48 out of 160) of gliomas, respectively. Its expression was significantly correlated with histological type of the tumours; however, no significant association between the expression of the ligand and its receptor was observed. The absence of PDGFA expression was significantly associated with the age of patients and with poor prognosis. Although PDGFRA gene-activating mutations were not found, PDGFRA gene amplification was observed in 21.1% (12 out of 57) of gliomas. No association was found between the presence of PDGFRA gene amplification and expression, excepting for grade II diffuse astrocytomas. CONCLUSION: The concurrent expression of PDGFA and PDGFRA in different subtypes of gliomas, reinforce the recognised significance of this signalling pathway in gliomas. PDGFRA gene amplification rather than gene mutation may be the underlying genetic mechanism driving PDGFRA overexpression in a portion of gliomas. Taken together, our results could provide in the future a molecular basis for PDGFRA-targeted therapies in gliomas., OM is a recipient of a PhD fellowship (SFRH/BD/36463/2007) from Fundação para a Ciência e a Tecnologia (FCT), Portugal. JSRF,MBKL, AM, NT, KF and AA are funded by Breakthrough BreastCancer. This study was supported in part by Pfizer/Sociedade de Ciências dicas de Lisboa with the prize: Research in oncology diseases, Professor Francisco Gentil’ and Breakthrough Breast Cancer., info:eu-repo/semantics/publishedVersion

Aims: Gastrointestinal stromal tumours (GISTs) are commonly driven by oncogenic mutations in KIT and PDGFRA. However, 10-40% of these patients are wild-type for these genes. The prognostic significance of wild-type GISTs is controversial, and they rarely respond to imatinib. The aim of this study was to elucidate the molecular lesions underlying wild-type GISTs tumorigenesis. Methods and results: Twenty-nine KIT and PDGFRA wild-type GISTs were re-assessed for the presence of 'cryptic'KIT exon 11 duplications. Using a specific polymerase chain reaction assay, three previously undetected mutations were identified. In the remaining 26 wild-type GISTs, KIT, stem cell factor (SCF), phospho-KIT and phospho-ERK expression was evaluated by immunohistochemistry. Samples were screened for gain-of-function mutations in the mitogen-activated protein kinase (MAPK) cascade. KIT and SCF co-expression associated with KIT activation was observed in approximately 30% of cases. Furthermore, phospho-ERK expression showed that MAPK is activated in approximately 30% of cases. None of RAS family (H-, K- and N-RAS) oncogenes exhibited activating mutations, whereas BRAF mutations were found in approximately 4% of cases. Conclusions: In the absence of RAS mutations, MAPK could be activated through SCF/KIT autocrine/paracrine mechanisms and/or mutated BRAF in a subset of KIT/PDGFRA wild-type GISTs., O.M. is the recipient of a PhD fellowship (SFRH ⁄ BD ⁄ 36463 ⁄ 2007) from FCT, Portugal. This study was partially supported by Novartis Oncology, Portugal.

Gliomas are the most common and devastating primary brain tumours. Despite therapeutic advances, the majority of gliomas do not respond either to chemo or radiotherapy. KIT, a class III receptor tyrosine kinase (RTK), is frequently involved in tumourigenic processes. Currently, KIT constitutes an attractive therapeutic target. In the present study we assessed the frequency of KIT overexpression in gliomas and investigated the genetic mechanisms underlying KIT overexpression. KIT (CD117) immunohistochemistry was performed in a series of 179 gliomas of various grades. KIT activating gene mutations (exons 9, 11, 13 and 17) and gene amplification analysis, as defined by chromogenic in situ hybridization (CISH) and quantitative real-time PCR (qRT-PCR) were performed in CD117 positive cases. Tumour cell immunopositivity was detected in 15.6% (28/179) of cases, namely in 25% (1/4) of pilocytic astrocytomas, 25% (5/20) of diffuse astrocytomas, 20% (1/5) of anaplastic astrocytomas, 19.5% (15/77) of glioblastomas and one third (3/9) of anaplastic oligoastrocytomas. Only 5.7% (2/35) of anaplastic oligodendrogliomas showed CD117 immunoreactivity. No association was found between tumour CD117 overexpression and patient survival. In addition, we also observed CD117 overexpression in endothelial cells, which varied from 0-22.2% of cases, being more frequent in high-grade lesions. No KIT activating mutations were identified. Interestingly, CISH and/or qRT-PCR analysis revealed the presence of KIT gene amplification in 6 glioblastomas and 2 anaplastic oligoastrocytomas, corresponding to 33% (8/24) of CD117 positive cases. In conclusion, our results demonstrate that KIT gene amplification rather than gene mutation is a common genetic mechanism underlying KIT expression in subset of malignant gliomas. Further studies are warranted to determine whether glioma patients exhibiting KIT overexpression and KIT gene amplification may benefit from therapy with anti-KIT RTK inhibitors., FCT(SFRH/BI/15257/2004). This work was partially supported by NOVARTIS Oncology, Portugal, and Breakthrough Breast Cancer, UK.

Receptor tyrosine kinase (RTK) targeted therapy has been explored for glioblastoma treatment. However, it is unclear which RTK inhibitors are the most effective and there are no predictive biomarkers available. We recently identified the RTK AXL as a putative target for the pan-RTK inhibitors cediranib and sunitinib, which are under clinical trials for glioblastoma patients. Here, we provide evidence that AXL activity can modulate sunitinib response in glioblastoma cell lines. We found that AXL knockdown conferred lower sensitivity to sunitinib by rescuing migratory defects and inhibiting apoptosis in cells expressing high AXL basal levels. Accordingly, overactivation of AXL by its ligand GAS6 rendered AXL positive glioblastoma cells more sensitive to sunitinib. AXL knockdown induced a cellular rewiring of several growth signaling pathways through activation of RTKs, such as EGFR, as well as intracellular pathways such as MAPK and AKT. The combination of sunitinib with a specific AKT inhibitor reverted the resistance of AXL-silenced cells to sunitinib. Together, our results suggest that sunitinib inhibits AXL and AXL activation status modulates therapy response of glioblastoma cells to sunitinib. Moreover, it indicates that combining sunitinib therapy with AKT pathway inhibitors could overcome sunitinib resistance., This work was funded by Fundacao para a Ciencia e Tecnologia (FCT), Portugal (project: PTDC/SAU-TOX/114549/2009). Olga Martinho is a recipient of a Post-Doc fellowship (UMINHO/BPD/32/2013) from QREN. We would like to acknowledge Dr. Shuang-En Chuang from the National Health Research Institute, Taiwan, for providing AXL vectors, and Dr. Raquel Andrade for critical review of the manuscript.

Epidermal growth factor receptor (EGFR) is overexpressed in up to 90% of head and neck squamous cell carcinoma (HNSCC) tumors. Cetuximab is the first targeted (anti-EGFR) therapy approved for the treatment of patients with locally advanced HNSCC. However, its efficacy is limited due to primary and secondary resistance, and there is no predict biomarkers of response. New generation of EGFR inhibitors with pan HER targeting and irreversible action, such as afatinib and allitinib represents a significant therapeutic promise in HNSCC treatment. In this study, we intend to compare the potential cytotoxicity of two irreversible anti-EGFR inhibitors (afatinib and allitinib) with cetuximab and to identify potential predictive biomarkers of response in a panel of HNSCC cell lines. The mutational analysis in hotspot regions of EGFR, KRAS, NRAS, PIK3CA and PTEN in the eight HNSCC cell lines revealed an EGFR mutation (p.H773Y) and gene amplification in the HN13 cells, and KRAS mutation in the JHU-28 cell line. According to the growth inhibition score (GI), allitinib was the most cytotoxic drug, followed by afatinib and finally cetuximab. The HN13 cell line exhibited a less responsive behavior to all drugs assessed, and interestingly, also displayed the higher levels of AKT phosphorylation. Therefore, we further performed drug combinations and found that with anti-AKT agent (MK2206), afatinib and allitinib, but not cetuximab, sensitivity was restored. Additionally, AKT1gene editing in afatinib and allitinib-treated cell lines showed that AKT1 silencing-induced cell line decreased viability and increased cytotoxicity through caspases 3/7activation.Additionally, in silico analysis of TCGA database showed that AKT1 overexpression was present in 14.7% (41/279) of HNSCC cases, and was associated with perineural invasion in advanced stage. In conclusion, allitinib presented a greater cytotoxic profile when compared to afatinib and cetuximab, and the AKT pathway constitutes a predictive marker of allitinib response. Importantly, in allitinib and afatinib-resistant cases, the pharmacological combination with AKT inhibitors could restore response and increase treatment success. Citation Format: Renato José Silva-Oliveira, Matias Melendez, Olga Martinho, Maicon Fernando Zanon, Luciano de Souza Viana, André Lopes Carvalho, Rui Manuel Reis. AKT can modulate the in vitro response of HNSCC cell to irreversible EGFR inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4091. doi:10.1158/1538-7445.AM2017-4091