The role of TNF-α in chordoma progression and inflammatory pathways Abstract Purpose Chordomas are highly therapy-resistant primary bone tumors that exhibit high relapse rates and may induce local destruction. Here, we evaluated the effects of tumor necrosis factor-alpha (TNF-α) on chordoma progression and clinical outcome. Methods Chordoma cells were treated with TNF-α after which its short- and long-term effects were evaluated. Functional assays, qRT-PCR and microarray-based expression analyses were carried out to assess the effect of TNF-α on chemo-resistance, epithelial to mesenchymal transition (EMT), migration, invasion and cancer stem cell-like properties. Finally, relationships between TNF-α expression and clinicopathological features were assessed in a chordoma patient cohort. Results We found that TNF-α treatment increased the migration and invasion of chordoma cells. Also, NF-κB activation was observed along with increased EMT marker expression. In addition, enhanced tumor sphere formation and soft agar colony formation were observed, concomitantly with increased chemo-resistance and CD338 marker expression. The TNF-α and TNFR1 expression levels were found to be significantly correlated with LIF, PD-L1 and Ki67 expression levels, tumor volume and a short survival time in patients. In addition, a high neutrophil to lymphocyte ratio was found to be associated with recurrence and a decreased overall survival. Conclusions From our data we conclude that TNF-α may serve as a prognostic marker for chordoma progression and that tumor-promoting inflammation may be a major factor in chordoma tumor progression.

Interplay between HSF1 and p53 signaling pathways in cancer initiation and progression: non-oncogene and oncogene addiction Abstract Background The p53 and HSF1 transcription factors are key players in cellular responses to stress. They activate important signaling pathways triggering adaptive mechanisms that maintain cellular homeostasis. HSF1 is mainly activated by proteotoxic stress, and its induction leads to the synthesis of chaperones that provide proteome integrity. The p53 protein, which is primarily activated in response to DNA damage, causes cell cycle arrest allowing for DNA repair or directs cells to apoptosis, thereby maintaining genome integrity. Both signaling pathways are also involved in neoplastic transformation and tumor progression. Loss of tumor suppressor abilities of the wild-type p53 protein results in oncogenesis, whereas proper HSF1 action, though non-oncogenic itself, actively supports this process. Conclusions Here, we describe in detail the interplay between the p53 and HSF1 signaling pathways, with particular emphasis on the molecular mechanisms involved, as well as their importance for normal cellular behavior, cancer development, the effectiveness of anti-cancer therapies and their toxicity. Detailed knowledge of the complex interplay between HSF1 and p53 may form a basis for the design of new protocols for cancer treatment.

Interplay between thyroid cancer cells and macrophages: effects on IL-32 mediated cell death and thyroid cancer cell migration Abstract Purpose Interleukin 32 (IL-32) is a pro-inflammatory cytokine of which different isoforms have been identified. Recently, IL-32 has been shown to act as a potent inducer of cell migration in several types of cancer. Although previous research showed that IL-32 is expressed in differentiated thyroid cancer (TC) cells, the role of IL-32 in TC cell migration has not been investigated. Furthermore, tumour-associated macrophages (TAMs) may play a facilitating role in cancer cell migration. The aim of this study was to explore whether the interaction between TC cells and TAMs results in increased expression of IL-32 in TC cells and to investigate whether this affects TC cell migration. Methods TPC-1 cells were co-culture with TC-induced or naive macrophages. Next, transcriptome analysis on TPC-1 cells was performed and supernatants were used for stimulation of TPC-1 cells. IL-32β and IL-32γ were exogenously overexpressed in TPC-1 cells using transient transfection, after which an in vitro gap closure assay was performed to assess cell migration, and the expression of migratory factors was assessed using RT-qPCR. Results We found that TC-induced macrophages induced IL-32 expression in TC cells and that TAM-derived TNFα was the main inducer of IL-32β expression in TC cells. Overexpression of IL-32β and IL-32γ did not affect TC cell migration, but increased cell death. Finally, we found that IL-32β overexpression led to increased mRNA expression of the pro-survival cytokine IL-8, while the expression of other migratory factors was not affected. Conclusions From our data, we conclude that TAM-derived TNFα induces IL-32β in TC cells. Although IL-32β does not affect TC cell migration, alternative splicing of IL-32 towards the IL-32β isoform may be beneficial for TC cell survival through induction of the pro-survival cytokine IL-8.

Clinical and biological impact of miR-18a expression in breast cancer after neoadjuvant chemotherapy Abstract Purpose The analysis of breast cancer residual tumors after neoadjuvant chemotherapy (nCT) may be useful for identifying new biomarkers. MicroRNAs are known to be involved in oncogenic pathways and treatment resistance of breast cancer. Our aim was to determine the role of miR-18a, a member of the miR-17-92a cluster, in breast cancer behavior and outcome after nCT. Methods Pre- and post-nCT tumor miR-18a expression was retrospectively assessed by qRT-PCR in 121 patients treated with nCT and was correlated with survival outcomes and with clinical and pathological characteristics. Breast cancer-derived MCF-7 and MDA-MB-231 cell lines were transfected with miR-18a and anti-miR-18a to evaluate the biological effects of this molecule. In addition, whole-transcriptome expression analysis was performed. Results High miR-18a expression in post-nCT residual tumors was found to be associated with a significantly worse overall survival [hazard ratio (HR): 2.80, 95% confidence interval (CI): 1.01–7.76] and a strong trend towards a poorer disease-free survival (HR: 2.44, 95% CI: 0.99–5.02) compared to low miR-18a expressing post-nCT residual tumors. Clinical and experimental data were found to be in conformity with the proliferative effects of miR-18a, which showed a significant correlation with Ki67 and MYBL2 expression, both in pre- and post-nCT tumors and in public databases. In vitro analysis of the role of miR-18a in breast cancer-derived cell lines showed that a high expression of miR-18a was associated with a low expression of the estrogen receptor (ER), a decreased sensitivity to tamoxifen and an enrichment in luminal B and endocrine resistance gene expression signatures. Conclusions From our data we conclude that post-nCT miR-18a expression in breast cancer serves as a negative prognostic marker, especially in luminal tumors. Clinical, in vitro and in silico data support the role of miR-18a in breast cancer cell proliferation and endocrine resistance and suggest its potential utility as a biomarker for additional adjuvant treatment in patients without a pathologic complete response to neoadjuvant therapy.

Apatinib inhibits glycolysis by suppressing the VEGFR2/AKT1/SOX5/GLUT4 signaling pathway in ovarian cancer cells Abstract Background Apatinib is a tyrosine kinase inhibitor that targets vascular endothelial growth factor receptor-2 (VEGFR2), and has shown encouraging therapeutic effects in various malignant tumors. As yet, however, the role of apatinib in ovarian cancer has remained unknown. Here, we sought to elucidate the role of apatinib in the in vitro and in vivo viability and proliferation of ovarian cancer cells, as well as in glucose metabolism in these cells. Methods The effects of apatinib on ovarian cancer cell viability and proliferation were assessed using Cell Counting Kit-8 (CCK-8) and colony formation assays, respectively. The expression of VEGFR2/AKT1/SOX5/GLUT4 pathway proteins was assessed using Western blotting, and glucose uptake and lactate production assays were used to detect glycolysis in ovarian cancer cells. SOX5 was exogenously over-expressed and silenced in ovarian cancer cells using expression vector and shRNA-based methods, respectively. RNA expression analyses were performed using RNA-seq and gene-chip-based methods. GLUT4 promoter activity was assessed using a dual-luciferase reporter assay. The expression of p-VEGFR2 (Tyr1175), p-AKT1 (Ser473), p-GSK3β (Ser9), SOX5 and GLUT4 in xenograft tissues was assessed using immunohistochemistry (IHC). Results We found that apatinib inhibited the in vitro and in vivo viability and proliferation in Hey and OVCA433 ovarian cancer cells in a dose-dependent and time-dependent manner. We also found that apatinib effectively suppressed glucose uptake and lactate production by blocking the expression of GLUT4 in these cells. In addition, we found that SOX5 predominantly rescued the inhibitory effect of apatinib on GLUT4 expression by activating its promoter. Finally, we found that apatinib regulated the expression of SOX5 by suppressing the VEGFR2/AKT1/GSK3β signaling pathway. Conclusions From our results, we conclude that apatinib suppresses the in vitro and in vivo viability and proliferation of ovarian cancer cells, as well as glycolysis by inhibiting the VEGFR2/AKT1/GSK3β/SOX5/GLUT4 signaling pathway. Apatinib may serve as a promising drug for the treatment of ovarian cancer.

Tumor-associated macrophages: role in cancer development and therapeutic implications Abstract Background Tumor-associated macrophages (TAMs) are known to play important roles in the initiation and progression of human cancers, as well as in angiogenesis. TAMs are considered as main components of the tumor microenvironment. Targeting TAMs may serve as a therapeutic strategy for the treatment of cancer. In this review, the signaling pathways, origin, function, polarization and clinical application of TAMs are discussed. The role of TAMs in tumor initiation, progression, angiogenesis, invasion and metastasis are also emphasized. In addition, a variety of clinical and pre-clinical approaches to target TAMs are discussed. Conclusions Clinical therapeutic approaches that show most promise include blocking the extravasation of TAMs along with using TAMs as diagnostic biomarkers for cancer progression. The targeting of TAMs in a variety of clinical settings appears to be a promising strategy for decreasing metastasis formation and for improving patient outcome.

Predictors of ribociclib-mediated antitumour effects in native and sorafenib-resistant human hepatocellular carcinoma cells Abstract Purpose The cyclin-dependent kinases (CDKs) CDK4 and CDK6 are important regulators of the cell cycle and represent promising targets in cancer treatment. We aimed to investigate the relevance of CDK4/6 in the development of hepatocellular carcinoma (HCC) and the potential of ribociclib, a novel orally available CDK4/6 inhibitor, as a treatment for HCC. Methods The effect of ribociclib was assessed in native and sorafenib-resistant HCC cell lines using viability assays, colony formation assays and FACS-based analyses. The expression of potential biomarkers of ribociclib response was assessed in cell lines and primary human hepatocytes using Western blotting. In addition, the prognostic relevance of the cyclin D-CDK4/6-retinoblastoma protein (Rb) pathway was assessed by analysing mRNA expression data from The Cancer Genome Atlas (TCGA). Results We found that ribociclib downregulated Rb and caused a profound loss of cell viability by inducing G1 cell cycle arrest in HCC cell lines exhibiting Rb-high/p16-low protein expression profiles, but not in Rb-low/p16-high cells, regardless their sensitivity to sorafenib. siRNA-based Rb silencing decreased cell proliferation, but did not diminish the sensitivity of HCC cells to ribociclib. Furthermore, we found that ribociclib synergized with sorafenib to cause cell death. mRNA analysis of primary human HCC specimens showed that CDK4 expression was correlated with patient survival and that the expression of Rb and the p16-encoding CDKN2A gene were inversely correlated. Conclusions From our data we conclude that impairment of the cyclin D-CDK4/6-Rb pathway is a frequent feature of HCC and that it is associated with a unfavourable prognosis. We also found that ribociclib exhibits a preferential antineoplastic activity in Rb-high HCC cells. Our results warrant further investigation of Rb and p16 expression as markers of HCC sensitivity to ribociclib.

Molecular profiling of anastatic cancer cells: potential role of the nuclear export pathway Abstract Purpose Anastasis is newly discovered process by which cells recover from late-stage apoptosis upon removal of a death stimulus. Recent reports suggest that cells may recover, even after the initiation of mitochondrial outer-membrane permeabilization (MOMP) and caspase activation. Here, we specifically studied the reversibility of late-stage apoptosis in cervical (HeLa) and breast (MDA-MB-231) cancer cells in relation to the extent of MOMP (limited or widespread). In addition, we explored the molecular factors involved in the anastatic process. Methods The extent of MOMP was assessed using time lapse confocal microscopic imaging, considering mitochondrial cytochrome c-GFP release as a marker for MOMP. Anastatic cells were generated by specifically recovering late-stage apoptotic (annexin V/PI positive) cervical and breast cancer cells. Molecular signaling events involved in death reversal were assessed using LC-MS/MS and qRT-PCR. Targeted chemical inhibition and shRNA-based gene silencing studies were employed to explore the role of the nuclear export pathway in anastasis and increased oncogenicity. Results Time-lapse imaging of drug-treated Cyt-c-GFP expressing cancer cells revealed cell recovery despite widespread MOMP. A few recovered anastatic cells were noted and these were found to proliferate through a selection-type of survival. They showed increased drug-resistance, migration and invasive potential compared to non-anastatic cancer cells. Network analysis using 49 proteins uniquely expressed in anastatic cells indicated upregulation of nuclear export/import, redox and Ras signaling pathways in both HeLa and MDA-MB-231 anastatic cells, indicating common molecular mechanisms in different cell types. Inhibition of XPO1 significantly reduced the recovery of apoptotic cells and abrogated acquired oncogenic transformation in the anastatic cancer cells. Conclusions Our study indicates that cancer cells can revert from apoptosis even after the induction of widespread MOMP. We noted a significant role of the nuclear-export pathway in the anastatic process of cancer cells. Inhibition of anastasis through the nuclear export pathway may be a potential therapeutic strategy for targeting drug-resistance, metastasis and recurrence problems during cancer treatment.

Recent advances in the clinical development of immune checkpoint blockade therapy Abstract Background The discovery of immune checkpoint proteins and the mechanisms by which cancer cells utilize them to evade the immune system has transformed our approach to cancer immunotherapy. Checkpoint blockade antibodies targeting cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death 1 (PD-1) and its ligands such as programmed cell death ligand 1 (PD-L1) have already revolutionized the treatment of multiple types of cancer and have significantly improved treatment and survival outcomes of patients affected by these malignancies. Conclusions Herein, we summarize current knowledge about the role of, and the mechanisms underlying PD-1/PD-L1 signaling pathways in antitumor immune responses, with particular emphasis on clinical studies evaluating the efficacy of anti-PD-1/PD-L1 blockade in various tumor types. Preliminary clinical investigations with immune-checkpoint blockers highlight broad opportunities with a high potential to enhance antitumor immunity and, as such, to generate significant clinical responses. These preliminary successes open up new avenues towards efficient therapeutics offered to patients.

Stromal organization as predictive biomarker for the treatment of colon cancer with adjuvant bevacizumab; a post-hoc analysis of the AVANT trial Abstract Purpose Intra-tumoral stroma has become increasingly important in understanding tumor biology, tumor progression and clinical outcome. The amount itself, quantified as the tumor-stroma ratio (TSR), has proven to be prognostic in stage I-III colon cancer. Also, alterations in stromal organization have been found to provide prognostic and predictive information in certain cancers. Here, we evaluated the predictive value of stromal organization in high-risk stage II and III colon cancer with respect to adjuvant bevacizumab and chemotherapy. Methods In a post-hoc analysis, stromal organization was microscopically determined in hematoxylin and eosin-stained primary tumor tissue samples of 1226 patients enrolled in the AVANT trial. Results We found that patients with tumors with a disorganized stroma showed different survival rates after the addition of bevacizumab compared to standard oxaliplatin-based chemotherapy regimens. However, overall this difference was not significant with a HR of 0.94 (95% CI 0.57–1.55; p = 0.80) for disease-free survival (DFS) and 1.01 (95% CI 0.51–1.99; p = 0.99) for overall survival (OS). Subgroup analysis, however, revealed that stromal organization combined with TSR allowed the identification of stroma-high patients with absolute cumulative survival benefits up to 15% when bevacizumab was added to oxaliplatin-based chemotherapy regimens. Conclusions In high-risk stage II and stage III colon cancer, we found that subgroup analysis of the combined parameters stromal organization and TSR allows for the identification of patients with absolute cumulative DFS and OS benefits of up to 15%, when adding bevacizumab to the currently recommended oxaliplatin-based chemotherapy. Stromal organization itself does, however, not serve as an independent prognostic or predictive parameter.