Melissa J. LaBonte

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.

Cancer dormancy: a model of early dissemination and late cancer recurrence

Cancer dormancy is a stage in tumor progression in which residual disease remains occult and asymptomatic for a prolonged period of time. Dormant tumor cells can be present as one of the earliest stages in tumor development, as well as a stage in micrometastases, and/or minimal residual disease left after an apparently successful treatment of the primary tumor. The general mechanisms that regulate the transition of disseminated tumor cells that have lain dormant into a proliferative state remain largely unknown. However, regulation of the growth from dormant tumor cells may be explained in part through the interaction of the tumor cell with its microenvironment, limitations in the blood supply, or an active immune system. An understanding of the regulatory machinery of these processes is essential for identifying early cancer biomarkers and could provide a rationale for the development of novel agents to target dormant tumor cells. This review focuses on the different signaling models responsible for early cancer dissemination and tumor recurrence that are involved in dormancy pathways. Clin Cancer Res; 18(3); 645–53. ©2011 AACR.

Molecular Pathways: Estrogen Pathway in Colorectal Cancer

Worldwide, colorectal cancer has a higher incidence rate in men than in women, suggesting a protective role for sex hormones in the development of the disease. Preclinical data support a role for estrogen and its receptors in the initiation and progression of colorectal cancer and establishes that protective effects of estrogen are exerted through ERβ. Hormone replacement therapy (HRT) in postmenopausal women as well as consumption of soy reduces the incidence of colorectal cancer. In the Womens Health Initiative trial, use of HRT in postmenopausal women reduced the risk of colon cancer by 56% [95% confidence interval (CI), 0.38–0.81; P = 0.003]. A recent meta-analysis showed that in women, consumption of soy reduced the risk of colon cancer by 21% (95% CI, 0.03–0.35; P = 0.026). In this review, using the preclinical data, we translate the findings in the clinical trials and observational studies to define the role of estrogen in the prevention of colorectal cancer. We hypothesize that sometime during the tumorigenesis process ERβ expression in colonocytes is lost and the estrogen ligand, HRT, or soy products, exerts its effects through preventing this loss. Thus, in the adenoma-to-carcinoma continuum, timing of HRT is a significant determinant of the observed benefit from this intervention. We further argue that the protective effects of estrogen are limited to certain molecular subtypes. Successful development of estrogen modulators for prevention of colorectal cancer depends on identification of susceptible colorectal cancer population(s). Thus, research to better understand the estrogen pathway is fundamental for clinical delivery of these agents. Clin Cancer Res; 19(21); 5842–8. ©2013 AACR.

Common cancer stem cell gene variants predict colon cancer recurrence

Purpose: Recent evidence suggests that cancer stem cells (CSC) are responsible for key elements of colon cancer progression and recurrence. Germline variants in CSC genes may result in altered gene function and/or activity, thereby causing interindividual differences in a patients tumor recurrence capacity and chemoresistance. We investigated germline polymorphisms in a comprehensive panel of CSC genes to predict time to tumor recurrence (TTR) in patients with stage III and high-risk stage II colon cancer. Experimental Design: A total of 234 patients treated with 5-fluorouracil–based chemotherapy at the University of Southern California were included in this study. Whole blood samples were analyzed for germline polymorphisms in genes that have been previously associated with colon CSC (CD44, Prominin-1, DPP4, EpCAM, ALCAM, Msi-1, ITGB1, CD24, LGR5, and ALDH1A1) by PCR-RFLP or direct DNA-sequencing. Results: The minor alleles of CD44 rs8193 C>T, ALCAM rs1157 G>A, and LGR5 rs17109924 T>C were significantly associated with increased TTR (9.4 vs. 5.4 years; HR, 0.51; 95% CI: 0.35–0.93; P = 0.022; 11.3 vs. 5.7 years; HR, 0.56; 95% CI: 0.33–0.94; P = 0.024, and 10.7 vs. 5.7 years; HR, 0.33; 95% CI: 0.12–0.90; P = 0.023, respectively) and remained significant in the multivariate analysis stratified by ethnicity. In recursive partitioning, a specific gene variant profile including LGR5 rs17109924, CD44 rs8193, and ALDH1A1 rs1342024 represented a high-risk subgroup with a median TTR of 1.7 years (HR, 6.71, 95% CI: 2.71–16.63, P < 0.001). Conclusion: This is the first study identifying common germline variants in colon CSC genes as independent prognostic markers for stage III and high-risk stage II colon cancer patients. Clin Cancer Res; 17(21); 6934–43. ©2011 AACR.

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.

Molecular markers in the treatment of metastatic colorectal cancer

Although significant progress has been made in colorectal cancer (CRC) treatment within the last decade with the approval of multiple new agents, the prognosis for patients with metastatic CRC remains poor with 5-year survival rates of ∼8%. Resistance to chemotherapy remains a major obstacle in effective CRC treatment and many patients do not receive any clinical benefit from chemotherapy. In addition, other patients will experience adverse reactions to treatment resulting in dose modifications or treatment withdrawal, which can severely reduce treatment efficacy. Currently, significant research efforts are attempting to identify reliable and validated biomarkers with which will guide clinicians to make more informed treatment decisions. Specifically, the use of molecular profiling has the potential to assist the clinician in administering the correct drug, dose, or intervention for the patient before the onset of therapy thereby selecting a treatment strategy likely to have the greatest clinical outcome while minimizing adverse events. However, until recently, personalized medicine is a paradigm that has existed more in conceptual terms than in reality with very few validated biomarkers used routinely in metastatic CRC treatment. Rapid advances in genomic, transcriptomic and proteomic technologies continues to improve our understanding of tumor biology, but the search for reliable biomarkers has turned out to be more challenging than previously anticipated with significant disparity in published literature and limited translation into routine clinical practice. Recent progress with the identification and validation of biomarkers to the anti-epidermal growth factor receptor monoclonal antibodies including KRAS and possibly BRAF provide optimism that the goal of individualized treatment is within reach. This review will highlight and discuss current progress in the search for biomarkers, the challenges this emerging field presents, and the future role of biomarkers in advancing CRC treatment.

Pharmacogenetic Angiogenesis Profiling for First-line Bevacizumab plus Oxaliplatin-Based Chemotherapy in Patients with Metastatic Colorectal Cancer

Purpose: There is substantial germline genetic variability within angiogenesis pathway genes, thereby causing interindividual differences in angiogenic capacity and resistance to antiangiogenesis therapy. We investigated germline polymorphisms in genes involved in VEGF-dependent and -independent angiogenesis pathways to predict clinical outcome and tumor response in metastatic colorectal cancer (mCRC) patients treated with bevacizumab and oxaliplatin-based chemotherapy. Experimental Design: A total of 132 patients treated with first-line bevacizumab and FOLFOX or XELOX were included in this study. Genomic DNA was isolated from whole-blood samples by PCR-RFLP or direct DNA sequencing. The endpoints of the study were progression-free survival (PFS), overall survival (OS), and response rate (RR). Results: The minor alleles of EGF rs444903 A>G and IGF-1 rs6220 A>G were associated with increased OS and remained significant in multivariate Cox regression analysis (HR: 0.52; 95% CI: 0.31–0.87; adjusted P = 0.012 and HR: 0.60; 95% CI: 0.36–0.99; adjusted P = 0.046, respectively). The minor allele of HIF1α rs11549465 C>T was significantly associated with increased PFS but lost its significance in multivariate analysis. CXCR1 rs2234671 G>C, CXCR2 rs2230054 T>C, EGFR rs2227983 G>A, and VEGFR-2 rs2305948 C>T predicted tumor response, with CXCR1 rs2234671 G>C remaining significant in multiple testing (Pact = 0.003). Conclusion: In this study, we identified common germline variants in VEGF-dependent and -independent angiogenesis genes predicting clinical outcome and tumor response in patients with mCRC receiving first-line bevacizumab and oxaliplatin-based chemotherapy. Clin Cancer Res; 17(17); 5783–92. ©2011 AACR.

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.