The growth of blood and lymphatic vasculature is mediated in part by secreted polypeptides of the vascular endothelial growth factor (VEGF) family. The prototype VEGF binds VEGF receptor (VEGFR)-1 ...and VEGFR-2 and is angiogenic, whereas VEGF-C, which binds to VEGFR-2 and VEGFR-3, is either angiogenic or lymphangiogenic in different assays. We used an adenoviral gene transfer approach to compare the effects of these growth factors in adult mice. Recombinant adenoviruses encoding human VEGF-C or VEGF were injected subcutaneously into C57Bl6 mice or into the ears of nude mice. Immunohistochemical analysis showed that VEGF-C upregulated VEGFR-2 and VEGFR-3 expression and VEGF upregulated VEGFR-2 expression at 4 days after injection. After 2 weeks, histochemical and immunohistochemical analysis, including staining for the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), the vascular endothelial marker platelet-endothelial cell adhesion molecule-1 (PECAM-1), and the proliferating cell nuclear antigen (PCNA) revealed that VEGF-C induced mainly lymphangiogenesis in contrast to VEGF, which induced only angiogenesis. These results have significant implications in the planning of gene therapy using these growth factors.
In the paracortex of lymph nodes, cellular immune responses are generated against antigens captured in peripheral tissues by dendritic cells (DCs). DC-SIGN (dendritic cell-specific ICAM-3 grabbing ...nonintegrin), a C-type lectin exclusively expressed by DCs, functions as an antigen receptor as well as an adhesion receptor. A functional homologue of DC-SIGN, L-SIGN (liver/lymph node-SIGN, also called DC-SIGN-related), is expressed by liver sinus endothelial cells. In lymph nodes, both DC-SIGN and L-SIGN are expressed. In this study, we analyzed the distribution of these two SIGN molecules in detail in both normal and immunoreactive lymph nodes. DC-SIGN is expressed by mature DCs in paracortical areas and in addition by DCs with an immature phenotype in the outer zones of the paracortex. L-SIGN expression was also detected in the outer zones on sinus endothelial cells characterized by their expression of the lymphatic endothelial markers LYVE-1 and CLEVER-1. During both cellular and humoral immune responses changes in the amount of DC-SIGN
+ immature and mature DCs and L-SIGN
+ endothelial cells were observed, indicating that the influx or proliferation of these cells is dynamically regulated.
CD44 has been implicated in tumor progression and metastasis, but the mechanism(s) involved is as yet poorly understood. Recent
studies have shown that CD44 isoforms containing the alternatively ...spliced exon v3 carry heparan sulfate side chains and are
able to bind heparin-binding growth factors. In the present study, we have explored the possibility of a physical and functional
interaction between CD44 and hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the receptor tyrosine kinase
c-Met. The HGF/SF-c-Met pathway mediates cell growth and motility and has been implicated in tumor invasion and metastasis.
We demonstrate that a CD44v3 splice variant efficiently binds HGF/SF via its heparan sulfate side chain. To address the functional
relevance of this interaction, Namalwa Burkittâs lymphoma cells were stably co-transfected with c-Met and either CD44v3 or
the isoform CD44s, which lacks heparan sulfate. We show that, as compared with CD44s, CD44v3 promotes: (i) HGF/SF-induced
phosphorylation of c-Met, (ii) phosphorylation of several downstream proteins, and (iii) activation of the MAP kinases ERK1
and -2. By heparitinase treatment and the use of a mutant HGF/SF with greatly decreased affinity for heparan sulfate, we show
that the enhancement of c-Met signal transduction induced by CD44v3 was critically dependent on heparan sulfate moieties.
Our results identify heparan sulfate-modified CD44 (CD44-HS) as a functional co-receptor for HGF/SF which promotes signaling
through the receptor tyrosine kinase c-Met, presumably by concentrating and presenting HGF/SF. As both CD44-HS and c-Met are
overexpressed on several types of tumors, we propose that the observed functional collaboration might be instrumental in promoting
tumor growth and metastasis.
To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA ...polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.
'Targeted' or 'biological' cancer treatments rely on differential gene expression between normal tissue and cancer, and genetic changes that render tumour cells especially sensitive to the agent ...being applied. Problems exist with the application of many agents as a result of damage to local tissues, tumour evolution and treatment resistance, or through systemic toxicity. Hence, there is a therapeutic need to uncover specific clinical targets which enhance the efficacy of cancer treatment whilst minimising the risk to healthy tissues. T-LAK cell-originated protein kinase (TOPK) is a MAPKK-like kinase which plays a role in cell cycle regulation and mitotic progression. As a consequence, TOPK expression is minimal in differentiated cells, although its overexpression is a pathophysiological feature of many tumours. Hence, TOPK has garnered interest as a cancer-specific biomarker and biochemical target with the potential to enhance cancer therapy whilst causing minimal harm to normal tissues. Small molecule inhibitors of TOPK have produced encouraging results as a stand-alone treatment in vitro and in vivo, and are expected to advance into clinical trials in the near future. In this review, we present the current literature pertaining to TOPK as a potential clinical target and describe the progress made in uncovering its role in tumour development. Firstly, we describe the functional role of TOPK as a pro-oncogenic kinase, followed by a discussion of its potential as a target for the treatment of cancers with high-TOPK expression. Next, we provide an overview of the current preclinical progress in TOPK inhibitor discovery and development, with respect to future adaptation for clinical use.
Abstract The ataxia telangiectasia and Rad3-related (ATR) plays an important role in maintaining genome integrity during DNA replication through the phosphorylation and activation of Chk1 and ...regulation of the DNA damage response. Preclinical studies have shown that disruption of ATR pathway can exacerbate the levels of replication stress in oncogene-driven murine tumors to promote cell killing. Additionally, inhibition of ATR can sensitise tumor cells to radiation or chemotherapy. Accumulating evidence suggests that targeting ATR can selectively sensitize cancer cells but not normal cells to DNA damage. Furthermore, in hypoxic conditions, ATR blockade results in overloading replication stress and DNA damage response causing cell death. Despite the attractiveness of ATR inhibition in the treatment of cancer, specific ATR inhibitors have remained elusive. In the last two years however, selective ATR inhibitors suitable for in vitro and – most recently – in vivo studies have been identified. In this article, we will review the literature on ATR function, its role in DDR and the potential of ATR inhibition to enhance the efficacy of radiation and chemotherapy.
Tumor hypoxia fuels an aggressive tumor phenotype and confers resistance to anticancer treatments. We conducted a clinical trial to determine whether the antimalarial drug atovaquone, a known ...mitochondrial inhibitor, reduces hypoxia in non-small cell lung cancer (NSCLC).
Patients with NSCLC scheduled for surgery were recruited sequentially into two cohorts: cohort 1 received oral atovaquone at the standard clinical dose of 750 mg twice daily, while cohort 2 did not. Primary imaging endpoint was change in tumor hypoxic volume (HV) measured by hypoxia PET-CT. Intercohort comparison of hypoxia gene expression signatures using RNA sequencing from resected tumors was performed.
Thirty patients were evaluable for hypoxia PET-CT analysis, 15 per cohort. Median treatment duration was 12 days. Eleven (73.3%) atovaquone-treated patients had meaningful HV reduction, with median change -28% 95% confidence interval (CI), -58.2 to -4.4. In contrast, median change in untreated patients was +15.5% (95% CI, -6.5 to 35.5). Linear regression estimated the expected mean HV was 55% (95% CI, 24%-74%) lower in cohort 1 compared with cohort 2 (
= 0.004), adjusting for cohort, tumor volume, and baseline HV. A key pharmacodynamics endpoint was reduction in hypoxia-regulated genes, which were significantly downregulated in atovaquone-treated tumors. Data from multiple additional measures of tumor hypoxia and perfusion are presented. No atovaquone-related adverse events were reported.
This is the first clinical evidence that targeting tumor mitochondrial metabolism can reduce hypoxia and produce relevant antitumor effects at the mRNA level. Repurposing atovaquone for this purpose may improve treatment outcomes for NSCLC.
To investigate mitophagy in 5 patients with severe dominantly inherited optic atrophy (DOA), caused by depletion of OPA1 (a protein that is essential for mitochondrial fusion), compared with healthy ...controls.
Patients with severe DOA (DOA plus) had peripheral neuropathy, cognitive regression, and epilepsy in addition to loss of vision. We quantified mitophagy in dermal fibroblasts, using 2 high throughput imaging systems, by visualizing colocalization of mitochondrial fragments with engulfing autophagosomes.
Fibroblasts from 3 biallelic OPA1(-/-) patients with severe DOA had increased mitochondrial fragmentation and mitochondrial DNA (mtDNA)-depleted cells due to decreased levels of OPA1 protein. Similarly, in siRNA-treated control fibroblasts, profound OPA1 knockdown caused mitochondrial fragmentation, loss of mtDNA, impaired mitochondrial function, and mitochondrial mislocalization. Compared to controls, basal mitophagy (abundance of autophagosomes colocalizing with mitochondria) was increased in (1) biallelic patients, (2) monoallelic patients with DOA plus, and (3) OPA1 siRNA-treated control cultures. Mitophagic flux was also increased. Genetic knockdown of the mitophagy protein ATG7 confirmed this by eliminating differences between patient and control fibroblasts.
We demonstrated increased mitophagy and excessive mitochondrial fragmentation in primary human cultures associated with DOA plus due to biallelic OPA1 mutations. We previously found that increased mitophagy (mitochondrial recycling) was associated with visual loss in another mitochondrial optic neuropathy, Leber hereditary optic neuropathy (LHON). Combined with our LHON findings, this implicates excessive mitochondrial fragmentation, dysregulated mitophagy, and impaired response to energetic stress in the pathogenesis of mitochondrial optic neuropathies, potentially linked with mitochondrial mislocalization and mtDNA depletion.
Background
Enhancer of zeste 2 (EZH2) promotes prostate cancer progression. We hypothesized that increased EZH2 expression is associated with postradiotherapy metastatic disease recurrence, and may ...promote radioresistance.
Methods
EZH2 expression was investigated using immunohistochemistry in diagnostic prostate biopsies of 113 prostate cancer patients treated with radiotherapy with curative intent. Associations between EZH2 expression in malignant and benign tissue in prostate biopsy cores and outcomes were investigated using univariate and multivariate Cox regression analyses. LNCaP and PC3 cell radiosensitivity was investigated using colony formation and γH2AX assays following UNC1999 chemical probe‐mediated EZH2 inhibition.
Results
While there was no significant association between EZH2 expression and biochemical recurrence following radiotherapy, univariate analysis revealed that prostate cancer cytoplasmic and total EZH2 expression were significantly associated with metastasis development postradiotherapy (P = 0.034 and P = 0.003, respectively). On multivariate analysis, the prostate cancer total EZH2 expression score remained statistically significant (P = 0.003), while cytoplasmic EZH2 expression did not reach statistical significance (P = 0.053). No association was observed between normal adjacent prostate EZH2 expression and biochemical recurrence or metastasis. LNCaP and PC3 cell treatment with UNC1999 reduced histone H3 lysine 27 tri‐methylation levels. Irradiation of LNCaP or PC3 cells with a single 2 Gy fraction with UNC1999‐mediated EZH2 inhibition resulted in a statistically significant, though modest, reduction in cell colony number for both cell lines. Increased γH2AX foci were observed 24 hours after ionizing irradiation in LNCaP cells, but not in PC3, following UNC1999‐mediated EZH2 inhibition vs controls.
Conclusions
Taken together, these results reveal that high pretreatment EZH2 expression in prostate cancer in diagnostic biopsies is associated with an increased risk of postradiotherapy metastatic disease recurrence, but EZH2 function may only at most play a modest role in promoting prostate cancer cell radioresistance.
Pre-clinically, phosphoinositide 3-kinase (PI3K) inhibition radiosensitises tumours by increasing intrinsic radiosensitivity and by reducing tumour hypoxia. We assessed whether buparlisib, a class 1 ...PI3K inhibitor, can be safely combined with radiotherapy in patients with non-small cell lung carcinoma (NSCLC) and investigated its effect on tumour hypoxia.
This was a 3 + 3 dose escalation and dose expansion phase I trial in patients with advanced NSCLC. Buparlisib dose levels were 50 mg, 80 mg and 100 mg once daily orally for 2 weeks, with palliative thoracic radiotherapy (20 Gy in 5 fractions) delivered during week 2. Tumour hypoxic volume (HV) was measured using 18F-fluoromisonidazole positron-emission tomography–computed tomography at baseline and following 1 week of buparlisib.
Twenty-one patients were recruited with 9 patients evaluable for maximum tolerated dose (MTD) analysis. No dose-limiting toxicity was reported; therefore, 100 mg was declared the MTD, and 10 patients received this dose in the expansion phase. Ninety-four percent of treatment-related adverse events were ≤grade 2 with fatigue (67%), nausea (24%) and decreased appetite (19%) most common per patient. One serious adverse event (grade 3 hypoalbuminaemia) was possibly related to buparlisib. No unexpected radiotherapy toxicity was reported. Ten (67%) of 15 patients evaluable for imaging analysis were responders with 20% median reduction in HV at the MTD.
This is the first clinical trial to combine a PI3K inhibitor with radiotherapy in NSCLC and investigate the effects of PI3K inhibition on tumour hypoxia. This combination was well tolerated and PI3K inhibition reduced hypoxia, warranting investigation into whether this novel class of radiosensitisers can improve radiotherapy outcomes.
•Buparlisib, a phosphoinositide 3-kinase (PI3K) inhibitor, is safe when combined with thoracic radiotherapy.•PI3K inhibition resulted in a rapid reduction in tumour hypoxia in non–small cell lung carcinoma.•This study supports the development of PI3K inhibitors as novel radiosensitisers.
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