Robert D. Ladner

Interleukin‐8 is associated with proliferation, migration, angiogenesis and chemosensitivity in vitro and in vivo in colon cancer cell line models

Interleukin‐8 (IL‐8), a chemokine with a defining CXC amino acid motif, is known to possess tumorigenic and proangiogenic properties. Overexpression of IL‐8 has been detected in many human tumors, including colorectal cancer (CRC), and is associated with poor prognosis. The goal of our study was to determine the role of IL‐8 overexpression in CRC cells in vitro and in vivo. We stably transfected the IL‐8 cDNA into two human colon cancer cell lines, HCT116 and Caco2, and selected IL‐8‐secreting transfectants. Real‐time RT‐PCR confirmed that IL‐8 mRNA was overexpressed in IL‐8 transfectants with 45‐ to 85‐fold higher than parental cells. The IL‐8‐transfected clones secreted 19‐ to 28‐fold more IL‐8 protein than control and parental cells as detected by ELISA. The IL‐8 transfectants demonstrated increased cellular proliferation, cell migration and invasion based on functional assays. Growth inhibition studies showed that IL‐8 overexpression lead to a significant resistance to oxaliplatin (p < 0.0001). Inhibition of IL‐8 overexpression with small interfering RNA reversed the observed increases in tumorigenic functions and oxaliplatin resistance, suggesting that IL‐8 not only provides a proliferative advantage but also promotes the metastatic potential of colon cancer cells. Using a tumor xenograft model, IL‐8‐expressing cells formed significantly larger tumors than the control cells with increased microvessel density. Together, these findings indicate that overexpression of IL‐8 promotes tumor growth, metastasis, chemoresistance and angiogenesis, implying IL‐8 to be an important therapeutic target in CRC.

Interleukin-8 and its receptor CXCR2 in the tumour microenvironment promote colon cancer growth, progression and metastasis

Background:Colorectal cancer (CRC) is a leading cause of death in the United States. Increased level of interleukin-8 (IL-8) and CXCR2 on tumours and in the tumour microenvironment has been associated with CRC growth, progression and recurrence in patients. Here, we aimed to evaluate the effects of tissue microenvironment-encoded IL-8 and CXCR2 on colon cancer progression and metastasis.Methods:A novel immunodeficient, skin-specific IL-8-expressing transgenic model was generated to evaluate colon cancer growth and metastasis. Syngeneic mouse colon cancer cells were grafted in CXCR2 knockout (KO) mice to study the contribution of CXCR2 in the microenvironment to cancer growth.Results:Elevated levels of IL-8 in the serum and tumour microenvironment profoundly enhanced the growth of human and mouse colon cancer cells with increased peri-tumoural angiogenesis, and also promoted the extravasation of the cancer cells into the lung and liver. The tumour growth was inhibited in CXCR2 KO mice with significantly reduced tumour angiogenesis and increased tumour necrosis.Conclusion:Increased expression of IL-8 in the tumour microenvironment enhanced colon cancer growth and metastasis. Moreover, the absence of its receptor CXCR2 in the tumour microenvironment prevented colon cancer cell growth. Together, our study demonstrates the critical roles of the tumour microenvironment-encoded IL-8/CXCR2 in colon cancer pathogenesis, validating the pathway as an important therapeutic target.

Standing the test of time: targeting thymidylate biosynthesis in cancer therapy

Over the past 60 years, chemotherapeutic agents that target thymidylate biosynthesis and the enzyme thymidylate synthase (TS) have remained among the most-successful drugs used in the treatment of cancer. Fluoropyrimidines, such as 5-fluorouracil and capecitabine, and antifolates, such as methotrexate and pemetrexed, induce a state of thymidylate deficiency and imbalances in the nucleotide pool that impair DNA replication and repair. TS-targeted agents are used to treat numerous solid and haematological malignancies, either alone or as foundational therapeutics in combination treatment regimens. We overview the pivotal discoveries that led to the rational development of thymidylate biosynthesis as a chemotherapeutic target, and highlight the crucial contribution of these advances to driving and accelerating drug development in the earliest era of cancer chemotherapy. The function of TS as well as the mechanisms and consequences of inhibition of this enzyme by structurally diverse classes of drugs with distinct mechanisms of action are also discussed. In addition, breakthroughs relating to TS-targeted therapies that transformed the clinical landscape in some of the most-difficult-to-treat cancers, such as pancreatic, colorectal and non-small-cell lung cancer, are highlighted. Finally, new therapeutic agents and novel mechanism-based strategies that promise to further exploit the vulnerabilities and target resistance mechanisms within the thymidylate biosynthesis pathway are reviewed.

Polymorphisms in Cyclooxygenase-2 and Epidermal Growth Factor Receptor Are Associated with Progression-Free Survival Independent of K-ras in Metastatic Colorectal Cancer Patients Treated with Single-Agent Cetuximab

Purpose: Recently, an objective response rate of 12% was reported in a phase II study of cetuximab in patients with epidermal growth factor receptor (EGFR)-expressing metastatic colorectal cancer (mCRC) refractory to fluoropyrimidine-, oxaliplatin-, and irinotecan-based chemotherapy (IMC-0144). In this large molecular correlates study, we tested whether K-ras mutation status and polymorphisms in genes involved in the EGFR-signaling pathway were associated with clinical outcome in IMC-0144. Experimental Design: We analyzed all available tissue samples from 130 of 346 mCRC patients enrolled in the IMC-0144 phase II clinical trial of cetuximab. Genomic DNA was extracted from formalin-fixed paraffin-embedded tumor tissues, and K-ras mutation status and the genotypes were analyzed using PCR-RFLP, direct DNA-sequencing, and 5′-end [γ-33P] ATP–labeled PCR-protocols. Results: The PFS of patients with cyclooxygenase-2 (COX-2) −765 G>C [C/C; risk ratio (RR), 0.31; 95% confidence interval (95% CI), 0.12-0.84; P = 0.032], COX-2 +8473 T>C (C/C; RR, 0.67; 95% CI, 0.40-1.13; P = 0.003), EGF +61 A>G (G/G; RR, 0.57; 95% CI, 0.34-0.95; P = 0.042), and EGFR +497 G>A (A/G; RR, 0.82; 95% CI, 0.56-1.20; P = 0.017) genotypes was significantly longer compared with those with other genotypes. In addition, patients whose tumors did not have K-ras mutations showed better RR, PFS, and overall survival than patients with K-ras mutations. In multivariable analysis, COX-2 +8473 T>C (adjusted P = 0.013) and EGFR +497 G>A (adjusted P = 0.010) remained significantly associated with progression-free survival, independent of skin rash toxicity, K-ras mutation status, and Eastern Cooperative Group performance status. Conclusions: Polymorphisms in COX-2 and EGFR may be useful independent molecular markers to predict clinical outcome in patients with mCRC treated with single-agent cetuximab, independent of skin rash toxicity, K-ras mutation, and Eastern Cooperative Oncology Group performance status.

Preferential Induction of EphB4 over EphB2 and Its Implication in Colorectal Cancer Progression

The receptor tyrosine kinase EphB2 is expressed by colon progenitor cells; however, only 39% of colorectal tumors express EphB2 and expression levels decline with disease progression. Conversely, EphB4 is absent in normal colon but is expressed in all 102 colorectal cancer specimens analyzed, and its expression level correlates with higher tumor stage and grade. Both EphB4 and EphB2 are regulated by the Wnt pathway, the activation of which is critically required for the progression of colorectal cancer. Differential usage of transcriptional coactivator cyclic AMP-responsive element binding protein-binding protein (CBP) over p300 by the Wnt/beta-catenin pathway is known to suppress differentiation and increase proliferation. We show that the beta-catenin-CBP complex induces EphB4 and represses EphB2, in contrast to the beta-catenin-p300 complex. Gain of EphB4 provides survival advantage to tumor cells and resistance to innate tumor necrosis factor-related apoptosis-inducing ligand-mediated cell death. Knockdown of EphB4 inhibits tumor growth and metastases. Our work is the first to show that EphB4 is preferentially induced in colorectal cancer, in contrast to EphB2, whereby tumor cells acquire a survival advantage.

The Human dUTPase Gene Encodes both Nuclear and Mitochondrial Isoforms DIFFERENTIAL EXPRESSION OF THE ISOFORMS AND CHARACTERIZATION OF A cDNA ENCODING THE MITOCHONDRIAL SPECIES

We have previously identified distinct nuclear and mitochondrial isoforms of dUTPase in human cells, reporting the cDNA sequence of the nuclear isoform (DUT-N). We now report a cDNA corresponding to the mitochondrial isoform (DUT-M). The DUT-M cDNA contains an 252-amino acid open reading frame, encoding a protein with a predicted M r of 26,704. The amino-terminal region of the protein contains an arginine-rich, 69-residue mitochondrial targeting presequence that is absent in the mature protein. In vitro transcription and translation of the DUT-M cDNA results in the production of a precursor protein with an apparent molecular mass of 31 kDa as judged by SDS-polyacrylamide gel electrophoresis. The DUT-M precursor is enzymatically active and immunoreacts with a dUTPase-specific monoclonal antibody. Mitochondrial import and processing studies demonstrate that the DUT-M precursor is processed into a 23-kDa protein and imported into mitochondria in vitro. Isoelectric focusing experiments demonstrate that the DUT-N has a pI of 6.0, while the processed form of DUT-M has a more basic pI of 8.1, measurements that are in agreement with predicted values. Studies aimed at understanding the expression of these isoforms were performed utilizing quiescent and replicating 34Lu human lung fibroblasts as a model cell culture system. Northern blot analysis, employing an isoform-specific probe, demonstrates that DUT-N and DUT-M are encoded by two distinct mRNA species of 1.1 and 1.4 kilobases, respectively. Western and Northern blot analysis reveal that DUT-M protein and mRNA are expressed in a constitutive fashion, independent of cell cycle phase or proliferation status. In contrast, DUT-N protein and mRNA levels are tightly regulated to coincide with nuclear DNA replication status. Because DUT-N and DUT-M have identical amino acid and cDNA sequences in their overlapping regions, we set out to determine if they were encoded by the same gene. The 5′ region of the gene encoding dUTPase was isolated and characterized by a combination of Southern hybridization and DNA sequencing. These analyses demonstrate that the dUTPase isoforms are encoded by the same gene with isoform-specific transcripts arising through the use of alternative 5′ exons. This finding represents the first example in humans of alternative 5′ exon usage to generate differentially expressed nuclear and mitochondrial specific protein isoforms.

DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

BackgroundDespite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes.MethodsHCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity® Pathway Analysis.ResultsTreatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed.ConclusionThis study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing much-needed information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer.

Thymidylate Synthase Haplotype is Associated with Tumor Recurrence in Stage II and Stage III Colon Cancer Patients

Background Tumor recurrence after curative resection is a major problem in the management of colon cancer therapy. Identifying molecular markers for tumor recurrence is critical for successfully selecting patients who are more likely to benefit from adjuvant chemotherapy. We analyzed the value of thymidylate synthase (TS) and methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms as a prognostic marker in stage II and stage III colon cancer patients treated with 5-fluorouracil-based adjuvant chemotherapy. Methods Between 1987 and 2007, blood samples were obtained from 197 patients with stage II or stage III colon cancer at medical facilities at the University of Southern California. DNA was extracted from peripheral blood, and the genotypes were analyzed using PCR–restriction fragment length polymorphism technique. Results Patients harboring the TS 3RG/+6-bp haplotype were at greatest risk to develop tumor recurrence [relative risk (RR): 2.25; 95% confidence interval (CI): 1.04–4.85; adjusted P value=0.032]. TS enhancer region 3RG alone (RR: 3.48 years; 95% CI: 1.61–7.54; adjusted P value=0.013) or in combination with TS 1494del6 bp (RR: 3.41 years; 95% CI: 1.33–8.75; adjusted P value=0.044) proved to be adverse prognostic markers in both univariate and multivariable analysis. Conclusion ‘High-expression’ variants of TS 2R/3R repeat, TS enhancer region 3R G/C, TS 1494del6 bp, and TS haplotype analysis might help to identify stage II and stage III colon cancer patients who are at great risk of developing tumor recurrence, and also those who are more likely to benefit from 5-fluorouracil-based adjuvant chemotherapy. Larger, independent, prospective studies are, however, needed to confirm and validate our preliminary findings.

The dual EGFR/HER2 inhibitor lapatinib synergistically enhances the antitumor activity of the histone deacetylase inhibitor panobinostat in colorectal cancer models.

As key molecules that drive progression and chemoresistance in gastrointestinal cancers, epidermal growth factor receptor (EGFR) and HER2 have become efficacious drug targets in this setting. Lapatinib is an EGFR/HER2 kinase inhibitor suppressing signaling through the RAS/RAF/MEK (MAP/ERK kinase)/MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase)/AKT pathways. Histone deacetylase inhibitors (HDACi) are a novel class of agents that induce cell cycle arrest and apoptosis following the acetylation of histone and nonhistone proteins modulating gene expression and disrupting HSP90 function inducing the degradation of EGFR-pathway client proteins. This study sought to evaluate the therapeutic potential of combining lapatinib with the HDACi panobinostat in colorectal cancer (CRC) cell lines with varying EGFR/HER2 expression and KRAS/BRAF/PIK3CA mutations. Lapatinib and panobinostat exerted concentration-dependent antiproliferative effects in vitro (panobinostat range 7.2-30 nmol/L; lapatinib range 7.6-25.8 μmol/L). Combined lapatinib and panobinostat treatment interacted synergistically to inhibit the proliferation and colony formation in all CRC cell lines tested. Combination treatment resulted in rapid induction of apoptosis that coincided with increased DNA double-strand breaks, caspase-8 activation, and PARP cleavage. This was paralleled by decreased signaling through both the PI3K and MAPK pathways and increased downregulation of transcriptional targets including NF-κB1, IRAK1, and CCND1. Panobinostat treatment induced downregulation of EGFR, HER2, and HER3 mRNA and protein through transcriptional and posttranslational mechanisms. In the LoVo KRAS mutant CRC xenograft model, the combination showed greater antitumor activity than either agent alone, with no apparent increase in toxicity. Our results offer preclinical rationale warranting further clinical investigation combining HDACi with EGFR and HER2-targeted therapies for CRC treatment.

Histone deacetylase inhibitors suppress thymidylate synthase gene expression and synergize with the fluoropyrimidines in colon cancer cells

Despite recent therapeutic advances, the response rates to chemotherapy for patients with metastatic colon cancer remain at ∼50% with the fluoropyrimidine, 5‐fluorouracil (5‐FU), continuing to serve as the foundation chemotherapeutic agent for the treatment of this disease. Previous studies have demonstrated that overexpression of thymidylate synthase (TS) is a key determinant of resistance to 5‐FU‐based chemotherapy. Therefore, there is a significant need to develop alternative therapeutic strategies to overcome TS‐mediated resistance. In this study, we demonstrate that the histone deacetylase inhibitors (HDACi) vorinostat and LBH589 significantly downregulate TS gene expression in a panel of colon cancer cell lines. Downregulation of TS was independent of p53, p21 and HDAC2 expression and was achievable in vivo as demonstrated by mouse xenograft models. We provide evidence that HDACi treatment leads to a potent transcriptional repression of the TS gene. Combination of the fluoropyrimidines 5‐FU or FUdR with both vorinostat and LBH589 enhanced cell cycle arrest and growth inhibition. Importantly, the downstream effects of TS inhibition were significantly enhanced by this combination including the inhibition of acute TS induction and the enhanced accumulation of the cytotoxic nucleotide intermediate dUTP. These data demonstrate that HDACi repress TS expression at the level of transcription and provides the first evidence suggesting a direct mechanistic link between TS downregulation and the synergistic interaction observed between HDACi and 5‐FU. This study provides rationale for the continued clinical evaluation of HDACi in combination with 5‐FU‐based therapies as a strategy to overcome TS‐mediated resistance.