Over recent decades anti-angiogenic therapies (AATs) have produced promising results in the treatment of different malignancies. Unfortunately, resistance often develops and patients ultimately relapse. In an attempt to elucidate the causes of recurrence, most studies have focused on tumor responses to hypoxic conditions induced by AAT. However, strategies targeting these mechanisms of resistance are still failing to improve treatments. Furthermore, a potential direct impact of AAT on tumor cells cannot be overlooked. This review provides a novel overview of the different aspects of the tumor cell response to AAT. Conflicting data regarding the nature of this effect are discussed and reconciled, thus providing new insight into the discussion of tumor recurrence despite AAT.
The PTTG1-binding factor (PBF/PTTG1IP) has an emerging repertoire of roles, especially in thyroid biology, and functions as a protooncogene. High PBF expression is independently associated with poor prognosis and lower disease-specific survival in human thyroid cancer. However, the precise role of PBF in thyroid tumorigenesis is unclear. Here, we present extensive evidence demonstrating that PBF is a novel regulator of p53, a tumor suppressor protein with a key role in maintaining genetic stability, which is infrequently mutated in differentiated thyroid cancer. By coimmunoprecipitation and proximity-ligation assays, we show that PBF binds specifically to p53 in thyroid cells and significantly represses transactivation of responsive promoters. Further, we identify that PBF decreases p53 stability by enhancing ubiquitination, which appears dependent on the E3 ligase activity of Mdm2. Impaired p53 function was evident in a transgenic mouse model with thyroid-specific PBF overexpression (transgenic PBF mice), which had significantly increased genetic instability as indicated by fluorescent inter simple sequence repeat-PCR analysis. Consistent with this, approximately 40% of all DNA repair genes examined were repressed in transgenic PBF primary cultures, including genes with critical roles in maintaining genomic integrity such as Mgmt, Rad51, and Xrcc3. Our data also revealed that PBF induction resulted in up-regulation of the E2 enzyme Rad6 in murine thyrocytes and was associated with Rad6 expression in human thyroid tumors. Overall, this work provides novel insights into the role of the protooncogene PBF as a negative regulator of p53 function in thyroid tumorigenesis, in which PBF is generally overexpressed and p53 mutations are rare compared with other tumor types.
Pancreatic Neuroendocrine Neoplasms (pNEN) are rare tumors which treatment still represent an important clinical problem, due to the paucity of medical treatments. Due to tumor complexity, techniques as 3D cultures are important to study drug activity in a more realistic model. This study aims to compare three different 3D culture methods in order to understand which one can be considered the best option in terms of experimental easiness and reproducibility in studying the efficacy of a target drug on pNEN. The BON1 cell line was used as a pNEN model and the well-known Receptor Tyrosine Kinase inhibitor Sunitinib was used in order to better investigate the different features of each method. The investigated methods are: (1) 96-well hanging drop plates (HD plates), (2) 24-well plates with a cell-repellent surface, and (3) ultra-low attachment 96-well plates with clear round bottom (ULA plates). The evaluated parameters during the study were: cell seeding, easiness in spheroids formation, morphology, culture maintenance, medium change, spheroids monitoring, picture quality, spheroid perimeter measurement reproducibility error, possibility to perform assays into the seeding plate, overall time of the experiment. Moreover, we investigated how culture methods can influence experimental outcomes evaluating perimeter changes, cell viability and immunohistochemistry of spheroids treated with different Sunitinib concentrations. Results showed that each method has weak and strong points but, considering the easiness of spheroids maintenance and reproducibility results, ULA plates method appears to be the best approach to culture BON1 spheroids and, therefore, to study pNEN.
Bronchial carcinoids (BCs) are rare neuroendocrine tumors that are still orphans of medical treatment. Human BC primary cultures may display resistance to everolimus, an inhibitor of the mammalian target of rapamycin (mTOR), in terms of cell viability reduction. Our aim was to assess whether the novel dual phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor NVP-BEZ235 is effective in everolimus-resistant human BC tissues and cell lines. In addition, we searched for possible markers of the efficacy of mTOR inhibitors that may help in identifying the patients who may benefit from treatment with mTOR inhibitors, sparing them from ineffective therapy. We found that NVP-BEZ235 is twice as potent as everolimus in reducing cell viability and activating apoptosis in human BC tissues that display sensitivity to mTOR inhibitors, but is not effective in everolimus-resistant BC tissues and cell lines that bypass cyclin D1 downregulation and escape G0/G1 blockade. Rebound AKT activation was not observed in response to treatment with either mTOR inhibitor in the 'resistant' BC cells. In addition to total mTOR levels, putative markers of the sensitivity of BCs to mTOR inhibitors are represented by AKT, p70S6K (RPS6KB2), and ERK1/2 (MAPK3/1) protein levels. Finally, we validated these markers in an independent BC group. These data indicate that the dual PI3K/mTOR inhibitor NVP-BEZ235 is more potent than everolimus in reducing the proliferation of human BC cells. 'Resistant' cells display lower levels of mTOR, p70S6K, AKT, and ERK1/2, indicating that these proteins may be useful as predictive markers of resistance to mTOR and PI3K/mTOR inhibitors in human BCs.
The PTTG1-binding factor (PBF) is a transforming gene capable of eliciting tumor formation in xenograft models. However, the precise role of PBF in tumorigenesis and its prognostic value as a cancer biomarker remain largely uncharacterised, particularly in malignancies outside the thyroid. Here, we provide the first evidence that PBF represents a promising prognostic marker in colorectal cancer. Examination of a total of 39 patients demonstrated higher PBF expression at both the mRNA (P ¼ 0.009) and protein (P < 0.0001) level in colorectal tumors compared to matched normal tissue. Critically, PBF was most abundant in colorectal tumors associated with Extramural Vascular Invasion (EMVI), increased genetic instability (GI) and somatic TP53 mutations, all features linked with recurrence and poorer patient survival. We further demonstrate by glutathione-S-transferase (GST) pull-down and coimmunoprecipitation that PBF binds to the tumor suppressor protein p53, as well as to p53 mutants (D126-132, M133K, V197E, G245D, I255F and R273C) identified in the colorectal tumors. Importantly, overexpression of PBF in colorectal HCT116 cells interfered with the transcriptional activity of p53-responsive genes such as mdm2, p21 and sfn. Diminished p53 stability (> 90%; P < 0.01) was also evident with a concurrent increase in ubiquitinated p53. Human colorectal tumors with wild-type TP53 and high PBF expression also had low p53 protein levels (P < 0.05), further emphasizing a putative interaction between these genes in vivo. Overall, these results demonstrate an emerging role for PBF in colorectal tumorigenesis through regulating p53 activity, with implications for PBF as a prognostic indicator for invasive tumors.
Lemur tyrosine kinase 3 (LMTK3) is an oncogenic kinase that is involved in different types of cancer (breast, lung, gastric, colorectal) and biological processes including proliferation, invasion, migration, chromatin remodeling as well as innate and acquired endocrine resistance. However, the role of LMTK3 in response to cytotoxic chemotherapy has not been investigated thus far. Using both 2D and 3D tissue culture models, we found that overexpression of LMTK3 decreased the sensitivity of breast cancer cell lines to cytotoxic (doxorubicin) treatment. In a mouse model we showed that ectopic overexpression of LMTK3 decreases the efficacy of doxorubicin in reducing tumor growth. Interestingly, breast cancer cells overexpressing LMTK3 delayed the generation of double strand breaks (DSBs) after exposure to doxorubicin, as measured by the formation of γH2AX foci. This effect was at least partly mediated by decreased activity of ataxia-telangiectasia mutated kinase (ATM) as indicated by its reduced phosphorylation levels. In addition, our RNA-seq analyses showed that doxorubicin differentially regulated the expression of over 700 genes depending on LMTK3 protein expression levels. Furthermore, these genes were found to promote DNA repair, cell viability and tumorigenesis processes / pathways in LMTK3-overexpressing MCF7 cells. In human cancers, immunohistochemistry staining of LMTK3 in pre- and post-chemotherapy breast tumor pairs from four separate clinical cohorts revealed a significant increase of LMTK3 following both doxorubicin and docetaxel based chemotherapy. In aggregate, our findings show for the first time a contribution of LMTK3 in cytotoxic drug resistance in breast cancer.
Elucidating signaling driven by lemur tyrosine kinase 3 (LMTK3) could help drug development. Here, we solve the crystal structure of LMTK3 kinase domain to 2.1Å resolution, determine its consensus motif and phosphoproteome, unveiling in vitro and in vivo LMTK3 substrates. Via high-throughput homogeneous time-resolved fluorescence screen coupled with biochemical, cellular, and biophysical assays, we identify a potent LMTK3 small-molecule inhibitor (C28). Functional and mechanistic studies reveal LMTK3 is a heat shock protein 90 (HSP90) client protein, requiring HSP90 for folding and stability, while C28 promotes proteasome-mediated degradation of LMTK3. Pharmacologic inhibition of LMTK3 decreases proliferation of cancer cell lines in the NCI-60 panel, with a concomitant increase in apoptosis in breast cancer cells, recapitulating effects of LMTK3 gene silencing. Furthermore, LMTK3 inhibition reduces growth of xenograft and transgenic breast cancer mouse models without displaying systemic toxicity at effective doses. Our data reinforce LMTK3 as a druggable target for cancer therapy.
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