Although neoplastic cells exhibit relatively higher sensitivity to hyperthermia than normal cells, hyperthermia has had variable success as an anti-cancer therapy. This variable outcome might be due to the fact that cancer cells themselves have differential degrees of sensitivity to high temperature. We hypothesized that the varying sensitivity of colorectal cancer (CRC) cells to hyperthermia depends upon the differential induction of survival pathways. Screening of such pathways revealed that Extracellular Signal-Regulated Kinase (ERK) signaling is augmented by hyperthermia, and the extent of this modulation correlates with the mutation status of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS). Through clonal growth assays, apoptotic analyses and transcription reporter assays of CRC cells that differ only in KRAS mutation status we established that mutant KRAS cells are more sensitive to hyperthermia, as they exhibit sustained ERK signaling hyperactivation and increased Wingless/Integrated (WNT)/beta-catenin signaling. We propose that whereas increased levels of WNT and ERK signaling and a positive feedback between the two pathways is a major obstacle in anti-cancer therapy today, under hyperthermia the hyperinduction of the pathways and their positive crosstalk contribute to CRC cell death. Ascertaining the causative association between types of mutations and hyperthermia sensitivity may allow for a mutation profile-guided application of hyperthermia as an anti-cancer therapy. Since KRAS and WNT signaling mutations are prevalent in CRC, our results suggest that hyperthermia-based therapy might benefit a significant number, but not all, CRC patients.

Breast cancer is the most common cancer in women, with 1.7 million new cases each year. As early diagnosis and prognosis are crucial factors in cancer treatment, we investigated potential DNA methylation biomarkers of the tumour suppressor family Ras-association domain family (RASSF). Promoter hypermethylation of tumour suppressors leads to their inactivation and thereby promotes cancer development and progression. In this study we analysed the tumour suppressors RASSF1A and RASSF10. Our study shows that RASSF10 is expressed in normal breast but inactivated by methylation in breast cancer. We observed a significant inactivating promoter methylation of RASSF10 in primary breast tumours. RASSF10 is inactivated in 63% of primary breast cancer samples but only 4% of normal control breast tissue is methylated (p < 0.005). RASSF1A also shows high promoter methylation levels in breast cancer of 56% vs. 8% of normal tissue (p < 0.005). Interestingly more than 80% of breast cancer samples harboured a hypermethylation of RASSF10 and/or RASSF1A promoter. Matching samples exhibited a strong tumour specific promoter methylation of RASSF10 in comparison to the normal control breast tissue. Demethylation treatment of breast cancer cell lines MCF7 and T47D reversed RASSF10 promoter hypermethylation and re-established RASSF10 expression. In addition, we could show the growth inhibitory potential of RASSF10 in breast cancer cell lines MCF7 and T47D upon exogenous expression of RASSF10 by colony formation. We could further show, that RASSF10 induced apoptotic changes in MCF7 and T47D cells, which was verified by a significant increase in the apoptotic sub G1 fraction by 50% using flow cytometry for MCF7 cells. In summary, our study shows the breast tumour specific inactivation of RASSF10 and RASSF1A due to DNA methylation of their CpG island promoters. Furthermore RASSF10 was characterised by the ability to block growth of breast cancer cell lines by apoptosis induction.

T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) proteins (TCFs) from the High Mobility Group (HMG) box family act as the main downstream effectors of the Wnt signaling pathway. The mammalian TCF/LEF family comprises four nuclear factors designated TCF7, LEF1, TCF7L1, and TCF7L2 (also known as TCF1, LEF1, TCF3, and TCF4, respectively). The proteins display common structural features and are often expressed in overlapping patterns implying their redundancy. Such redundancy was indeed observed in gene targeting studies; however, individual family members also exhibit unique features that are not recapitulated by the related proteins. In the present viewpoint, we summarized our current knowledge about the specific features of individual TCFs, namely structural-functional studies, posttranslational modifications, interacting partners, and phenotypes obtained upon gene targeting in the mouse. In addition, we employed several publicly available databases and web tools to evaluate the expression patterns and production of gene-specific isoforms of the TCF/LEF family members in human cells and tissues.

Epigenetic silencing of RASSF1A is frequently observed in numerous cancers and has been previously reported. The promoter region of RASSF1A is predicted to have 75 CpG sites, and very few studies demonstrate how the methylation of these sites affects expression. In addition, the expression relationship between RASSF1A and its downstream target, modulator of apoptosis 1 (MOAP-1), is poorly understood. In this study, we have explored the mRNA expression of RASSF1A, MOAP-1 and the well-characterized splice variant of RASSF1, RASSF1C, in cancer cell lines and primary tumors. We confirmed that the RASSF1A promoter is robustly methylated within a 32-CpG region in solid tumors and results in lower mRNA expression. The MOAP-1 promoter contains ~110 CpG sites, but was not found to be methylated in cancer cell lines when 19 predicted CpG sites were explored. Interestingly, MOAP-1 mRNA expression positively correlated with RASSF1A expression in numerous cancers, whereas RASSF1C expression remained the same or was increased in cell lines or tissues with epigenetic loss of RASSF1A. We speculate that MOAP-1 and RASSF1A may be more intimately connected than originally thought, and the expression of both are warranted in experimental designs exploring the biology of the RASSF1A/MOAP-1 molecular pathway.

Pulmonary malignancies with neuroendocrine differentiation represent a rare subclass of lung carcinomas, which vary in the extent of differentiation and grade of biological aggressiveness. In particular, neuroendocrine tumors are classified into well differentiated typical and atypical carcinoids as well as poorly differentiated large cell neuroendocrine and small cell lung carcinomas. Tiny MicroRNAs have been identified as reliable classifiers in distinct cancer types and seem to play important roles in cellular processes like regulation of cell growth, differentiation and apoptosis. In the present study, two different microRNAs (miR-21 and miR-34a) were explored for their involvements in pathogenesis of subtypes and finally in differential diagnosis of pulmonary neuroendocrine tumors. miR-21 was upregulated in poorly differentiated neuroendocrine tumors (mean rank: 26.8; 28.75) as compared to carcinoids (mean rank: 12.33; 12.07) with a significance of 0.00033. High-expression levels of miR-34a were associated with atypical carcinoids (p = 0.010). A close association is implicated between the elevated miR-21 values in high-grade and miR-34a patterns in low-grade atypical neuroendocrine lung carcinomas, which could potentially be exploited as practical supportive markers for differential lung cancer diagnosis in routine. However, some additional extended research and validation studies are needed to utilize them as routine markers or potential molecular targets for personalized medicine.

Triple-negative breast cancers (TNBC) are characterized by aggressive tumour biology resulting in a poor prognosis. Androgen receptor (AR) is one of newly emerging biomarker in TNBC. In recent years, ARs have been demonstrated to play an important role in the genesis and in the development of breast cancer, although their prognostic role is still debated. In the present study, we explored the correlation of AR expression with clinical, pathological and molecular features and its impact on prognosis in early TNBC.

The relationship between obesity and breast cancer (BC) has focused on serum factors. However, the mammary gland contains adipose tissue (AT) which may enable the crosstalk between adipocytes and tumor cells contributing to tumor macrophage recruitment. We hypothesize that the breast AT (bAT) is inflamed in obese females and plays a major role in breast cancer development. The effects of this interplay on macrophage chemotaxis were examined in vitro, using co-cultures of mouse macrophages, mammary tumor cells and adipocytes. Macrophages were exposed to the adipocyte and tumor paracrine factors leptin, CCL2 and lauric acid (alone or in combinations). In cell supernatants Luminex identified additional molecules with chemotactic and other pro-tumor functions. Focus on the adipokine leptin, which has been shown to have a central role in breast cancer pathogenesis, indicated it modulates macrophage phenotypes and functions. In vivo experiments demonstrate that mammary tumors from obese mice are larger and that bAT from obese tumor-bearers contains higher numbers of macrophages/CLS and hypertrophic adipocytes than bAT from lean tumor-bearers, thus confirming it is more inflamed. Also, bAT distal from the tumor is more inflamed in obese than in lean mice. Our results reveal that bAT plays a role in breast cancer development in obesity.

Epidemiological studies provide strong evidence that obesity and the associated adipose tissue inflammation are risk factors for breast cancer; however, the molecular mechanisms are poorly understood. We evaluated the effect of a high-fat/high-calorie diet on mammary carcinogenesis in the immunocompetent MMTV-PyMT murine model. Four-week old female mice (20/group) were randomized to receive either a high-fat (HF; 60% kcal as fat) or a low-fat (LF; 16% kcal) diet for eight weeks. Body weights were determined, and tumor volumes measured by ultrasound, each week. At necropsy, the tumors and abdominal visceral fat were weighed and plasma collected. The primary mammary tumors, adjacent mammary fat, and lungs were preserved for histological and immunohistochemical examination and quantification of infiltrating macrophages, crown-like structure (CLS) formation, and microvessel density. The body weight gains, visceral fat weights, the primary mammary tumor growth rates and terminal weights, were all significantly greater in the HF-fed mice. Adipose tissue inflammation in the HF group was indicated by hepatic steatosis, pronounced macrophage infiltration and CLS formation, and elevations in plasma monocyte chemoattractant protein-1 (MCP-1), leptin and proinflammatory cytokine concentrations. HF intake was also associated with higher tumor-associated microvascular density and the proangiogenic factor MCP-1. This study provides preclinical evidence in a spontaneous model of breast cancer that mammary adipose tissue inflammation induced by diet, enhances the recruitment of macrophages and increases tumor vascular density suggesting a role for obesity in creating a microenvironment favorable for angiogenesis in the progression of breast cancer.

Exposure to stray neutrons increases the risk of second cancer development after proton therapy. Previously reported analytical models of this exposure were difficult to configure and had not been investigated below 100 MeV proton energy. The purposes of this study were to test an analytical model of neutron equivalent dose per therapeutic absorbed dose  at 75 MeV and to improve the model by reducing the number of configuration parameters and making it continuous in proton energy from 100 to 250 MeV. To develop the analytical model, we used previously published H/D values in water from Monte Carlo simulations of a general-purpose beamline for proton energies from 100 to 250 MeV. We also configured and tested the model on in-air neutron equivalent doses measured for a 75 MeV ocular beamline. Predicted H/D values from the analytical model and Monte Carlo agreed well from 100 to 250 MeV (10% average difference). Predicted H/D values from the analytical model also agreed well with measurements at 75 MeV (15% average difference). The results indicate that analytical models can give fast, reliable calculations of neutron exposure after proton therapy. This ability is absent in treatment planning systems but vital to second cancer risk estimation.

Colorectal cancer is a major global health problem resulting in over 600,000 deaths world-wide every year with the majority of these due to metastatic disease. Wnt signalling, and more specifically β-catenin-related transcription, has been shown to drive both tumorigenesis and the metastatic process in colorectal neoplasia, yet its complex interactions with other key signalling pathways, such as hedgehog, remain to be elucidated. We have previously shown that the Hedgehog (HH) signalling pathway is active in cells from colorectal tumours, and that inhibition of the pathway with cyclopamine induces apoptosis. We now show that cyclopamine treatment reduces β-catenin related transcription in colorectal cancer cell lines, and that this effect can be reversed by addition of Sonic Hedgehog protein. We also show that cyclopamine concomitantly induces expression of the tumour suppressor and prognostic indicator E-cadherin. Consistent with a role for HH in regulating the invasive potential we show that cyclopamine reduces the expression of transcription factors (Slug, Snail and Twist) associated with the epithelial-mesenchymal transition and reduces the invasiveness of colorectal cancer cells in vitro. Taken together, Cancers 2015, 7 1886 these data show that pharmacological inhibition of the hedgehog pathway has therapeutic potential in the treatment of colorectal cancer.

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States and incidence rates are rising. Both detection and treatment options for pancreatic cancer are limited, providing a less than 5% five-year survival advantage. The need for new biomarkers for early detection and treatment of pancreatic cancer demands the efficient translation of bench knowledge to provide clinical benefit. One source of therapeutic resistance is the pancreatic tumor microenvironment, which is characterized by desmoplasia and hypoxia making it less conducive to current therapies. A major factor regulating desmoplasia and subsequently promoting chemoresistance in pancreatic cancer is hepatocyte growth factor (HGF), the sole ligand for c-MET (mesenchymal-epithelial transition), an epithelial tyrosine kinase receptor. Binding of HGF to c-MET leads to receptor dimerization and autophosphorylation resulting in the activation of multiple cellular processes that support cancer progression. Inhibiting activation of c-MET in cancer cells, in combination with other approaches for reducing desmoplasia in the tumor microenvironment, might significantly improve the success of chemotherapy. Therefore, HGF makes a potent novel target for developing therapeutic strategies in combination with existing drugs for treating pancreatic adenocarcinoma. This review provides a comprehensive analysis of HGF and its promising potential as a chemotherapeutic target for pancreatic cancer.

A previous genome-wide screening analysis identified a panel of genes that sensitize the human non-small-cell lung carcinoma cell line NCI-H1155 to taxol. However, whether the identified genes sensitize other cancer cells to taxol has not been examined. Here, we silenced the taxol-sensitizer genes identified (acrbp, atp6v0d2, fgd4, hs6st2, psma6, and tubgcp2) in nine other cancer cell types (including lung, cervical, ovarian, and hepatocellular carcinoma cell lines) that showed reduced cell viability in the presence of a sub-lethal concentration of taxol. Surprisingly, none of the genes studied increased sensitivity to taxol in the tested panel of cell lines. As observed in H1155 cells, SKOV3 cells displayed induction of five of the six genes studied in response to a cell killing dose of taxol. The other cell types were much less responsive to taxol. Notably, four of the five inducible taxol-sensitizer genes tested (acrbp, atp6v0d2, psma6, and tubgcp2) were upregulated in a taxol-resistant ovarian cancer cell line. These results indicate that the previously identified taxol-sensitizer loci are not conserved genetic targets involved in inhibiting cell proliferation in response to taxol. Our findings also suggest that regulation of taxol-sensitizer genes by taxol may be critical for acquired cell resistance to the drug.

Children receiving radiotherapy face the probability of a subsequent malignant neoplasm (SMN). In some cases, the predicted SMN risk can be reduced by proton therapy. The purpose of this study was to apply the most comprehensive dose assessment methods to estimate the reduction in SMN risk after proton therapy vs. photon therapy for a 13-year-old girl requiring craniospinal irradiation (CSI). We reconstructed the equivalent dose throughout the patient's body from therapeutic and stray radiation and applied SMN incidence and mortality risk models for each modality. Excluding skin cancer, the risk of incidence after proton CSI was a third of that of photon CSI. The predicted absolute SMN risks were high. For photon CSI, the SMN incidence rates greater than 10% were for thyroid, non-melanoma skin, lung, colon, stomach, and other solid cancers, and for proton CSI they were non-melanoma skin, lung, and other solid cancers. In each setting, lung cancer accounted for half the risk of mortality. In conclusion, the predicted SMN risk for a 13-year-old girl undergoing proton CSI was reduced vs. photon CSI. This study demonstrates the feasibility of inter-institutional whole-body dose and risk assessments and also serves as a model for including risk estimation in personalized cancer care.

Human NAD(P)H quinone oxidoreductase-1 (hNQO1) is an important cancer-related biomarker, which shows significant overexpression in malignant cells. Developing an effective method for detecting NQO1 activity with high sensitivity and selectivity in tumors holds a great potential for cancer diagnosis, treatment, and management. In the present study, we report a new dicyanoisophorone (DCP) based fluorescent probe (NQ-DCP) capable of monitoring hNQO1 activity in vitro and in vivo in both ratiometric and turn-on model. NQ-DCP was prepared by conjugating dicyanoisophorone fluoroprobe with hNQO1 activatable quinone propionic acid (QPA), which remain non-fluorescent until activation by tumor-specific hNQO1. NQ-DCP featured a large Stokes shift (145 nm), excellent biocompatibility, cell permeability, and selectivity towards hNQO1 allowed to differentiate cancer cells from healthy cells. We have successfully employed NQ-DCP to monitor non-invasive endogenous hNQO1 activity in brain tumor cells in vitro and in xenografted tumors developed in nude mice.

The EphA3 receptor has recently emerged as a functional tumour-specific therapeutic target in glioblastoma (GBM). EphA3 is significantly elevated in recurrent disease, is most highly expressed on glioma stem cells (GSCs), and has a functional role in maintaining self-renewal and tumourigenesis. An unlabelled EphA3-targeting therapeutic antibody is currently under clinical assessment in recurrent GBM patients. In this study, we assessed the efficacy of EphA3 antibody drug conjugate (ADC) and radioimmunotherapy (RIT) approaches using orthotopic animal xenograft models. Brain uptake studies, using positron emission tomography/computed tomography (PET/CT) imaging, show EphA3 antibodies are effectively delivered across the blood-tumour barrier and accumulate at the tumour site with no observed normal brain reactivity. A robust anti-tumour response, with no toxicity, was observed using EphA3, ADC, and RIT approaches, leading to a significant increase in overall survival. Our current research provides evidence that GBM patients may benefit from pay-loaded EphA3 antibody therapies.

Liver-specific knockout of Nrf1 in the mouse leads to spontaneous development of non- alcoholic steatohepatitis with dyslipidemia, and then its deterioration results in hepatoma, but the underlying mechanism remains elusive to date. A similar pathological model is reconstructed here by using human Nrf1α-specific knockout cell lines. Our evidence has demonstrated that a marked increase of the inflammation marker COX2 definitely occurs in Nrf1α-/- cells. Loss of Nrf1α leads to hyperactivation of Nrf2, which results from substantial decreases in Keap1, PTEN and most of 26S proteasomal subunits in Nrf1α-/- cells. Further investigation of xenograft model mice showed that malignant growth of Nrf1α-/--derived tumors is almost abolished by silencing of Nrf2, while Nrf1α+/⁺-tumor is markedly repressed by an inactive mutant (i.e., Nrf2-/-ΔTA), but largely unaffected by a priori constitutive activator (i.e., caNrf2ΔN). Mechanistic studies, combined with transcriptomic sequencing, unraveled a panoramic view of opposing and unifying inter-regulatory cross-talks between Nrf1α and Nrf2 at different layers of the endogenous regulatory networks from multiple signaling towards differential expression profiling of target genes. Collectively, Nrf1α manifests a dominant tumor-suppressive effect by confining Nrf2 oncogenicity. Though as a tumor promoter, Nrf2 can also, in turn, directly activate the transcriptional expression of Nrf1 to form a negative feedback loop. In view of such mutual inter-regulation by between Nrf1α and Nrf2, it should thus be taken severe cautions to interpret the experimental results from loss of Nrf1α, Nrf2 or both.

Persistent infection with high-risk Human Papillomavirus (HR-HPV) is the main requisite for cervical cancer development. Normally, HPV is limited to the site of infection and regulates a plethora of cellular elements to avoid the immune surveillance by inducing an anti-inflammatory state, allowing the progress through the viral cycle and the carcinogenic process. Recent findings suggest that the AT-hook transcriptional factor AKNA could play a role in the development of cervical cancer. AKNA is strongly related to the expression of co-stimulatory molecules such CD40/CD40L to achieve an anti-tumoral immune response. To date, there is no evidence demonstrating the effect of the HPV E6 oncoprotein on the AT-hook factor AKNA. In this work, minimal expression of AKNA in cervical carcinoma compared to normal tissue was found. We show the ability of E6 from high-risk HPVs 16 and 18 to interact with and down-regulate AKNA as well as its co-stimulatory molecule CD40 in a proteasome dependent manner. We also found that p53 interacts with AKNA and promotes AKNA expression. Our results indicate that the de-regulation of CD40 and AKNA is induced by the HPV E6 oncoprotein, and this event involves the action of p53 suggesting that the axis E6/p53A/AKNA might play an important role in the de-regulation of the immune system during the carcinogenic process induced by HR-HPV.

Cancer cell proliferation and insufficient blood supply can lead to the development of hypoxic areas in the tumor tissue. The adaptation to the hypoxic environment is mediated by a transcriptional complex called hypoxia-inducible factor (HIF). HIF protein levels are tightly controlled by oxygen-dependent prolyl hydroxylase domain proteins (PHDs). However, the precise roles of these enzymes in tumor progression and their downstream signaling pathways are not fully characterized. Here, we study PHD3 function in murine experimental osteosarcoma. Unexpectedly, PHD3 silencing in LM8 cells affects neither HIF-1α protein levels, nor the expression of various HIF-1 target genes. Subcutaneous injection of PHD3-silenced tumor cells accelerated tumor progression and was accompanied by dramatic phenotypic changes in the tumor vasculature. Blood vessels in advanced PHD3-silenced tumors were enlarged whereas their density was greatly reduced. Examination of the molecular pathways underlying these alterations revealed that platelet-derived growth factor (PDGF)-C signaling is activated in the vasculature of PHD3-deficient tumors. Silencing of PDGF-C depleted tumor growth, increased vessel density and reduced vessel size. Our data show that PHD3 controls tumor growth and vessel architecture in LM8 osteosarcoma by regulating the PDGF-C pathway, and support the hypothesis that different members of the PHD family exert unique functions in tumors.

To evaluate the role of risk-adapted proton beam therapy (PBT) in hepatocellular carcinoma (HCC) patients, a total of 243 HCC patients receiving risk-adapted PBT with three dose-fractionation regimens (regimen A [n = 40], B [n = 60], and C [n = 143]) according to the proximity of their gastrointestinal organs (<1 cm, 1⁻1.9 cm, and ≥2 cm, respectively) were reviewed: The prescribed doses to planning target volume 1 (PTV1) were 50 gray equivalents (GyE) (EQD2 [equivalent dose in 2 Gy fractions], 62.5 GyE10), 60 GyE (EQD2, 80 GyE10), and 66 GyE (EQD2, 91.3 GyE10) in 10 fractions, respectively, and those of PTV2 were 30 GyE (EQD2, 32.5 GyE10) in 10 fractions. In all patients, the five-year local recurrence-free survival (LRFS) and overall survival (OS) rates were 87.5% and 48.1%, respectively, with grade ≥3 toxicity of 0.4%. In regimens A, B, and C, the five-year LRFS and OS rates were 54.6%, 94.7%, and 92.4% (p < 0.001), and 16.7%, 39.2%, and 67.9% (p < 0.001), respectively. The five-year OS rates of the patients with the Modified Union for International Cancer Control (mUICC) stages I, II, III, and IVA and Barcelona Clinic Liver Cancer (BCLC) stages A, B, and C were 69.2%, 65.4%, 43.8%, and 26.6% (p < 0.001), respectively, and 65.1%, 40%, and 32.2% (p < 0.001), respectively. PBT could achieve promising long-term tumor control and have a potential role as a complementary or alternative therapeutic option across all stages of HCC.

Targeting immune checkpoint molecules such as programmed death ligand-1 (PDL1) is an emerging strategy for anti-cancer therapy. However, transient expression of PDL1 and difficulty in tumor stroma penetration has limited the utility of anti-PDL1 therapy. To overcome these limitations, we report a new conjugate between the clinically approved PDL1 antibody (PDL1 AB) and drug Doxorubicin (Dox), named PDL1-Dox. We conjugated PDL1-Dox through a hydrazone linker containing a polyethylene glycol (PEG) spacer, which allows it to dissociate in a tumor environment and improves solubility. The purpose of using Dox is to disrupt the tumor extracellular environment so that PDL-1 antibody can penetrate the tumor core. PDL1-Dox demonstrates significant cell killing, disruption of tumor spheroid and induction of apoptosis in a breast cancer cell line. Significant release of IFN-γ suggests PDL1-Dox can upmodulate T cell activation. Optical imaging of dye conjugate supports the selective tumor targeting ability and core penetration of the construct.