[Show abstract][Hide abstract] ABSTRACT: The profound metabolic reprogramming that occurs in cancer cells has been investigated primarily in two-dimensional cell cultures, which fail to recapitulate spatial aspects of cell-to-cell interactions as well as tissue gradients present in three-dimensional tumours. Here, we describe an engineered model to assemble three-dimensional tumours by rolling a scaffold-tumour composite strip. By unrolling the strip, the model can be rapidly disassembled for snapshot analysis, allowing spatial mapping of cell metabolism in concert with cell phenotype. We also show that the establishment of oxygen gradients within samples that are shaped by oxygen-dependent signalling pathways, as well as the consequential variations in cell growth, response to hypoxic gradients extending from normoxia to severe hypoxia, and therapy responsiveness, are consistent with those of tumours in vivo. Moreover, by using liquid chromatography tandem mass spectrometry, we mapped cellular metabolism and identified spatially defined metabolic signatures of cancer cells to reveal both known and novel metabolic responses to hypoxia.
[Show abstract][Hide abstract] ABSTRACT: Purpose: Hypoxia constitutes an attractive therapeutic target owing to its prevalence in tumors and its contribution to disease progression and treatment resistance. Several hypoxia-activated prodrugs (HAPs) have been developed to address this target, including the Phase III candidate TH-302 (evofosfamide) and the preclinical agent SN30000, which is an optimized analogue of the well-studied HAP tirapazamine. Experience with this therapeutic class highlights an urgent need to identify biomarkers of HAP sensitivity, including enzymes responsible for prodrug activation during hypoxia.
Methods: We deployed whole-genome and reductase-focused lentiviral shRNA screens, in tandem with massively parallel sequencing, to identify determinants of sensitivity to SN30000 in hypoxic HCT116, HT-29 and PANC-1 cells. Sensitivity determinants were validated by RNAi-mediated knockdown and nuclease-mediated genetic knockout, and expression of potential predictive biomarkers interrogated by immunohistochemistry in three clinically-annotated cohorts of head and neck squamous cell carcinoma (HNSCC).
Results: Our functional screens identified the flavoprotein P450 (cytochrome) oxidoreductase (POR) as the predominant determinant of sensitivity to SN30000 in all cell lines evaluated, except a POR knockout clone derived from HCT116, with no other genes consistently selected. Specific hairpins against POR were validated by qPCR and immunoblotting, where POR suppression directly correlated with the degree of resistance conferred. Knockdown or knockout of POR inhibited metabolic activation of SN30000 and clonogenic killing of SN30000 and TH-302 under hypoxia. No other genes consistently modified SN30000 sensitivity, even within a POR-negative background. A retrospective evaluation of HNSCC showed a wide variation in POR expression and suggested a possible relationship with human papillomavirus status and HAP sensitivity.
Conclusions: Our study identifies POR as a potential predictive biomarker of HAP sensitivity that should be explored during clinical development of SN30000, TH-302, and other hypoxia directed agents.
[Show abstract][Hide abstract] ABSTRACT: Tumor-stroma interactions are regulated through a network of autocrine and
paracrine signaling pathways that are crucial for many aspects of tumor development.
Activation and signaling through the HGF/Met pathway have been shown to increase
cell motility and reduce sensitivity to chemotherapy. We used a patient-derived
colon-cancer epithelial stem cell line and patient-derived normal fibroblasts (NFs) or
cancer-associated fibroblasts (CAFs), chosen for high HGF expression, to investigate
the contribution of stromal signaling through the MET pathway in tumor growth and
sensitivity to the Met inhibitor crizotinib. Co-injection of the colon cancer cell line
with NFs or CAFs resulted in significantly increased tumor take. Additionally, tumors
derived from the co-injections showed increased growth rates, an invasion of host
stroma, increased expression of vimentin, and reduced sensitivity to crizotinib. These
data demonstrate that stromal signaling through HGF/Met from adjacent fibroblasts
leads to a more aggressive tumor phenotype, and can cause resistance to treatments
targeting this pathway.
[Show abstract][Hide abstract] ABSTRACT: The results from many studies indicate that most solid tumors, regardless of site of origin, contain hypoxic regions. Experimental studies have demonstrated that, apart from the well-known protective effect of hypoxia on the radiation response of cells and tissues, hypoxic conditions can also result in modified gene expression patterns, causing (to a greater or lesser extent in different cell populations) genomic instability, increased invasive capacity, higher propensity to metastasize, enhanced stem cell properties, and ability to survive nutrient deprivation. Clinical trials of hypoxia-targeted treatments have demonstrated improved local tumor control and patient survival in a number of tumor sites. However, our improved understanding of the underlying biology of cellular responses to hypoxia, and its potential interactions with the heterogeneous nature of tumor phenotypes, makes it likely that not every tumor that contains regions of hypoxia would necessarily need (or benefit from) such treatments. New more effective treatments are emerging, but it is likely that these treatments would have the biggest clinical effect in situations where tumor hypoxia is a primary driver of cancer behavior. The challenge for the Radiation Oncology community is the development of robust precision cancer medicine strategies for identifying patients with such tumors, in the setting of other etiological, genomic, and host-tumor factors, and treating these patients with the appropriate hypoxia-targeting strategy to reduce the effect of hypoxia on radiation treatment response. In this context, it is important to consider not only the hypoxic state of the tumor at diagnosis but also the changing characteristics of this state during the course of treatment.
No preview · Article · May 2015 · Seminars in radiation oncology
[Show abstract][Hide abstract] ABSTRACT: Although the MK3 gene was originally found deleted in some cancers, it is highly expressed in others. The relevance of MK3 for oncogenesis is currently not clear. We recently reported that MK3 controls ERK activity via a negative feedback mechanism. This prompted us to investigate a potential role for MK3 in cell proliferation. We here show that overexpression of MK3 induces a proliferative arrest in normal diploid human fibroblasts, characterized by enhanced expression of replication stress- and senescence-associated markers. Surprisingly, MK3 depletion evokes similar senescence characteristics in the fibroblast model. We previously identified MK3 as a binding partner of Polycomb Repressive Complex 1 (PRC1) proteins. In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus. In agreement with this, the PRC1 oncoprotein BMI1, but not the PCR2 protein EZH2, bypasses MK3-induced senescence in fibroblasts and suppresses P16INK4A expression. In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function. Notably, MK3 ablation enhances proliferation in two different cancer cells. Finally, the fibroblast model was used to evaluate the effect of potential tumorigenic MK3 driver-mutations on cell proliferation and M/SAPK signaling imbalance. Taken together, our findings support a role for MK3 in control of proliferation and replicative life-span, in part through concerted action with BMI1, and suggest that the effect of MK3 modulation or mutation on M/SAPK signaling and, ultimately, proliferation, is cell context-dependent.
[Show abstract][Hide abstract] ABSTRACT: Background:
Autophagy allows recycling of cellular components and may facilitate cell survival after chemotherapy. Pantoprazole inhibits proton pumps and is reported to inhibit autophagy. Here we evaluate the effects of pantoprazole to modify cytotoxicity of the anticancer drug docetaxel, and underlying mechanisms.
Effects of docetaxel±pantoprazole were studied against wild-type and autophagy-deficient PC3 cells and against four human xenografts. Effects of pantoprazole on autophagy were evaluated by quantifying LC3-I, LC3-II and p62 proteins in western blots, and by fluorescent microscopy of cells transfected with RFP-GFP-LC3. The distribution of drug effects and of autophagy was quantified in tumour sections in relation to blood vessels and hypoxia by immunohistochemistry using γH2AX, cleaved caspase-3, Ki67 and LC3/ p62.
Pantoprazole increased the toxicity of docetaxel in vitro, increased docetaxel-induced expression of γH2AX and cleaved caspase-3, and decreased Ki67 in tumour sections. Pantoprazole increased growth delay of four human xenografts of low, moderate and high sensitivity to docetaxel, with minimal increase in toxicity. Docetaxel led to increased autophagy throughout tumour sections. Pantoprazole inhibited autophagy, and effects of pantoprazole were reduced against genetically modified cells with decreased ability to undergo autophagy.
Autophagy is a mechanism of resistance to docetaxel chemotherapy that may be modified by pantoprazole to improve therapeutic index.
No preview · Article · Feb 2015 · British Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to determine the relative biological effectiveness (RBE) along the axis of two range-modulated proton beams (160 and 230 MeV). Both the depth and the dose dependence of RBE were investigated. Chinese hamster V79-WNRE cells, suspended in medium containing gelatin and cooled to 2°C, were used to obtain complete survival curves at multiple positions throughout the entrance and 10 cm spread-out Bragg peak (SOBP). Simultaneous measurements of the survival response to (60)Co gamma rays served as the reference data for the proton RBE determinations. For both beams the RBE increased significantly with depth in the 10 cm SOBP, particularly in the distal half of the SOBP, then rose even more sharply at the distal edge, the most distal position measured. At a 4 Gy dose of gamma radiation (S = 0.34) the average RBE values for the entrance, proximal half, distal half and distal edge were 1.07 ± 0.01, 1.10 ± 0.01, 1.17 ± 0.01 and 1.21 ± 0.01, respectively, and essentially the same for both beams. At a 2 Gy dose of gamma radiation (S = 0.71) the average RBE values rose to 1.13 ± 0.03, 1.15 ± 0.02, 1.26 ± 0.02 and 1.30 ± 0.02, respectively, for the same four regions of the SOBP. The difference between the 4 Gy and 2 Gy RBE values reflects the dose dependence of RBE as measured in these V79-WNRE cells, which have a low α/β value, as do other widely used cell lines that also show dose-dependent RBE values. Late-responding tissues are also characterized by low α/β values, so it is possible that these cell lines may be predictive for the response of such tissues (e.g., spinal cord, optic nerve, kidney, liver, lung). However, in the very small number of studies of late-responding tissues performed to date there appears to be no evidence of an increased RBE for protons at low doses. Similarly, RBE measurements using early responding in vivo systems (mostly mouse jejunum, an early-responding tissue which has a large α/β ∼10 Gy) have generally shown little or no detectable dose dependence. It is useful to compare the RBE values reported here to the commonly used generic clinical RBE of 1.1, which assumes no dependence on depth or on dose. Our proximal RBEs obviously avoid the depth-related increase in RBE and for doses of 4 Gy or more, the low-dose increase in RBE is also minimized, as shown in this article. Thus the proximal RBE at a 4 Gy dose of 1.10 ± 0.01, quoted above, represents an interesting point of congruence with the clinical RBE for conditions where it could reasonably be expected in the measurements reported here. The depth dependence of RBE reported here is consistent with the majority of measurements, both in vitro and in vivo, by other investigators. The dose dependence of RBE, on the other hand, is tissue specific but has not yet been demonstrated for protons by RBE values in late-responding normal tissue systems. This indicates a need for additional RBE determination as function of dose, especially in late-responding tissues.
No preview · Article · Jan 2015 · Radiation Research
[Show abstract][Hide abstract] ABSTRACT: Background: Hypoxia constitutes an attractive therapeutic target owing to its prevalence in tumours and its contribution to disease progression and treatment resistance. Several hypoxia-selective prodrugs have been developed to address this target, including the preclinical agent SN30000 which is an optimised analogue of the well-studied prodrug tirapazamine. These prodrugs are enzymatically activated under hypoxic conditions and thus selectively toxic to viable hypoxic cells. Biomarkers that are predictive of prodrug activation and tumour sensitivity are urgently needed to support their clinical development. Materials and methods: We deployed whole-genome lentiviral shRNA screens interrogating 82,017 unique hairpins targeted to 16,019 human genes, and a high-representation focused library enriched for oxidoreductases (1,821 hairpins targeting 359 genes), in tandem with massively parallel sequencing, to identify determinants of sensitivity to SN30000 in HCT116, HT-29 and PANC-1 cells under hypoxic conditions. Potential determinants were evaluated by RNAi-mediated knockdown and zinc finger nuclease-mediated genetic knockout in cell lines and xenografts. Results: Our functional screens identified the flavoprotein P450 (cytochrome) oxidoreductase (POR) as the predominant determinant of sensitivity to SN30000 in all cell lines evaluated, except a POR knockout clone derived from HCT116, with no other genes consistently selected. Specific hairpins against POR were validated by qPCR and immunoblotting, where POR suppression directly correlated with the degree of enrichment in the high-throughput screens. Knockdown or knockout of POR inhibited metabolic activation of SN30000 and enhanced clonogenic survival of cells exposed under hypoxic conditions. We are currently assessing effects of POR knockout on the activity of SN30000 against hypoxic cells in xenografts. Conclusions: POR acts as a critical determinant of tumour cell sensitivity to SN30000 by catalysing reductive activation of the prodrug. As a single gene apparently responsible for a major component of SN30000 activity, tumour expression of POR may be a clinically tractable predictive marker of response to SN30000. The methodology established in this study may be repurposed to study determinants of sensitivity to additional hypoxia-activated prodrugs such as the clinical agents TH-302 and PR-104.
[Show abstract][Hide abstract] ABSTRACT: Targeted blockade of aberrantly activated signaling pathways is an attractive therapeutic strategy for solid tumors, but drug resistance is common. KRAS is a frequently mutated gene in human cancer but remains a challenging clinical target. Inhibitors against KRAS signaling mediators, namely, PI3K (phosphatidylinositol 3-kinase) and mTOR (mechanistic target of rapamycin), have limited clinical efficacy as single agents in KRAS-mutant colorectal cancer (CRC). We investigated potential bypass mechanisms to PI3K/mTOR inhibition in KRAS-mutant CRC. Using genetically engineered mouse model cells that had acquired resistance to the dual PI3K/mTOR small-molecule inhibitor PF-04691502, we determined with chemical library screens that inhibitors of the ERBB [epidermal growth factor receptor (EGFR)] family restored the sensitivity to PF-04691502. Although EGFR inhibitors alone have limited efficacy in reducing KRAS-mutant tumors, we found that PF-04691502 induced the abundance, phosphorylation, and activity of EGFR, ERBB2, and ERBB3 through activation of FOXO3a (forkhead box O 3a), a transcription factor inhibited by the PI3K to AKT pathway. PF-04691502 also induced a stem cell-like gene expression signature. KRAS-mutant patient-derived xenografts from mice treated with PF-04691502 had a similar gene expression signature and exhibited increased EGFR activation, suggesting that this drug-induced resistance mechanism may occur in patients. Combination therapy with dacomitinib (a pan-ERBB inhibitor) restored sensitivity to PF-04691502 in drug-resistant cells in culture and induced tumor regression in drug-resistant allografts in mice. Our findings suggest that combining PI3K/mTOR and EGFR inhibitors may improve therapeutic outcome in patients with KRAS-mutant CRC.
No preview · Article · Nov 2014 · Science Signaling
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs are small regulatory RNAs that post transcriptionally control gene expression. Reduced expression of DICER, the enzyme involved in microRNA processing, is frequently observed in cancer and is associated with poor clinical outcome in various malignancies. Yet, the underlying mechanisms are not well understood. Here we identify tumour hypoxia as a regulator of DICER expression in large cohorts of breast cancer patients. We show that DICER expression is suppressed by hypoxia through an epigenetic mechanism that involves inhibition of oxygen-dependent H3K27me3 demethylases KDM6A/B and results in silencing of the DICER promoter. Subsequently, reduced miRNA processing leads to derepression of the miR-200 target ZEB1, stimulates the epithelial to mesenchymal transition and ultimately results in the acquisition of stem cell phenotypes in human mammary epithelial cells. Our study uncovers a previously unknown relationship between oxygen-sensitive epigenetic regulators, miRNA biogenesis and tumour stem cell phenotypes that may underlie poor outcome in breast cancer.
Full-text · Article · Oct 2014 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: Cancer-related deregulation of miRNA biogenesis has been suggested, but the underlying mechanisms remain elusive. Here we report a previously unrecognized effect of hypoxia in the downregulation of Drosha and Dicer in cancer cells that leads to dysregulation of miRNA biogenesis and increased tumour progression. We show that hypoxia-mediated downregulation of Drosha is dependent on ETS1/ELK1 transcription factors. Moreover, mature miRNA array and deep sequencing studies reveal altered miRNA maturation in cells under hypoxic conditions. At a functional level, this phenomenon results in increased cancer progression in vitro and in vivo, and data from patient samples are suggestive of miRNA biogenesis downregulation in hypoxic tumours. Rescue of Drosha by siRNAs targeting ETS1/ELK1 in vivo results in significant tumour regression. These findings provide a new link in the mechanistic understanding of global miRNA downregulation in the tumour microenvironment.
Full-text · Article · Oct 2014 · Nature Communications