Sustained inhibition of deacetylases is required for the antitumor activity of the histone deactylase inhibitors panobinostat and vorinostat in models of colorectal cancer

Despite compelling preclinical data in colorectal cancer (CRC), the efficacy of HDACIs has been disappointing in the clinic. The goal of this study was to evaluate the effectiveness of vorinostat and panobinostat in a dose- and exposure-dependent manner in order to better understand the dynamics of drug action and antitumor efficacy. In a standard 72 h drug exposure MTS assay, notable concentration-dependent antiproliferative effects were observed in the IC50 range of 1.2-2.8 μmol/L for vorinostat and 5.1-17.5 nmol/L for panobinostat. However, shorter clinically relevant exposures of 3 or 6 h failed to elicit any significant growth inhibition and in most cases a >24 h exposure to vorinostat or panobinostat was required to induce a sigmoidal dose-response. Similar results were observed in colony formation assays where ≥ 24 h of exposure was required to effectively reduce colony formation. Induction of acetyl-H3, acetyl-H4 and p21 by vorinostat were transient and rapidly reversed within 12 h of drug removal. In contrast, panobinostat-induced acetyl-H3, acetyl-H4, and p21 persisted for 48 h after an initial 3 h exposure. Treatment of HCT116 xenografts with panobinostat induced significant increases in acetyl-H3 and downregulation of thymidylate synthase after treatment. Although HDACIs exert both potent growth inhibition and cytotoxic effects when CRC cells were exposed to drug for ≥ 24 h, these cells demonstrate an inherent ability to survive HDACI concentrations and exposure times that exceed those clinically achievable. Continued efforts to develop novel HDACIs with improved pharmacokinetics/phamacodynamics, enhanced intratumoral delivery and class/isoform-specificity are needed to improve the therapeutic potential of HDACIs and HDACI-based combination regimens in solid tumors.

Assessing the in vivo efficacy of biologic antiangiogenic therapies

PURPOSE: To review key clinical issues underlying the assessment of in vivo efficacy when using antiangiogenic therapies for cancer treatment.

METHODS: Literature relevant to use of antiangiogenic therapies in cancer was reviewed, with particular emphasis on the assessment of in vivo efficacy of these agents, as well as additional angiogenic factors that could play a role in escape from angiogenesis inhibition.

RESULTS: In order to grow and metastasize, tumors need to continually acquire new blood supplies; therefore, therapeutic inhibition of angiogenesis has become a component of anticancer treatment for many tumor types. Bevacizumab, a humanized monoclonal antibody directed at vascular endothelial growth factor A (VEGF-A), has shown activity in combination with chemotherapy in metastatic colorectal cancer. Nevertheless, the use of antiangiogenic therapies remains suboptimal; specifically, optimal dose, duration of therapy, and combination of agents remain unknown. Also, at present, it is not possible to determine which patients are most likely to respond to a given form of antiangiogenic therapy. There has been increased recognition of alternative pathways possibly associated with disease progression in patients undergoing antiangiogenic therapy targeted at VEGF-A. Multiligand-targeted antiangiogenic therapies, such as ziv-aflibercept (formerly known as aflibercept, VEGF Trap), are currently undergoing clinical evaluation. Ziv-aflibercept forms monomeric complexes with VEGF-A, VEGF-B, and PlGF, which have a long half-life, allowing optimization of ziv-aflibercept doses and angiogenic blockage.

CONCLUSIONS: Although antiangiogenic therapies have increased treatment options for cancer patients, their use is limited by a lack of established and standardized methodology to evaluate their efficacy in vivo. Circulating endothelial cells, hypertension, and several molecular and imaging-based markers have potential for use as biomarkers in these patients and may better define appropriate patient populations.

A phase I/II trial of vorinostat in combination with 5-fluorouracil in patients with metastatic colorectal cancer who previously failed 5-FU-based chemotherapy

PURPOSE: We conducted a phase I/II clinical trial to determine the safety and feasibility of combining vorinostat with 5-fluorouracil (5-FU) in patients with metastatic colorectal cancer (mCRC) and elevated intratumoral thymidylate synthase (TS).

METHODS: Patients with mCRC who had failed all standard therapeutic options were eligible. Intratumoral TS mRNA expression and peripheral blood mononuclear cell (PBMC) histone acetylation were measured before and after 6 consecutive days of vorinostat treatment at 400 mg PO daily. 5-FU/LV were given on days 6 and 7 and repeated every 2 weeks, along with continuous daily vorinostat. Dose escalation occurred in cohorts of three to six patients.

RESULTS: Ten patients were enrolled. Three dose levels were explored in the phase I portion of the study. Two dose-limiting toxicities (DLTs) were observed at the starting dose level, which resulted in dose de-escalation to levels -1 and -2. Given the occurrence of two DLTs at each of the dose levels, we were unable to establish a maximum tolerated dose (MTD). Two patients achieved significant disease stabilization for 4 and 6 months. Grade 3 and 4 toxicities included fatigue, thrombocytopenia and mucositis. Intratumoral TS downregulation > or = 50% was observed in one patient only. Acetylation of histone 3 was observed in PBMCs following vorinostat treatment.

CONCLUSIONS: The study failed to establish a MTD and was terminated. The presence of PBMC histone acetylation indicates biological activity of vorinostat, however, consistent reductions in intratumoral TS mRNA were not observed. Alternate vorinostat dose-scheduling may alleviate the toxicity and achieve optimal TS downregulation.

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.

Novel opportunities for thymidylate metabolism as a therapeutic target

For over 40 years, the fluoropyrimidine 5-fluorouracil (5-FU) has remained the central agent in therapeutic regimens employed in the treatment of colorectal cancer and is frequently combined with the DNA-damaging agents oxaliplatin and irinotecan, increasing response rates and improving overall survival. However, many patients will derive little or no benefit from treatment, highlighting the need to identify novel therapeutic targets to improve the efficacy of current 5-FU-based chemotherapeutic strategies. dUTP nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi, providing substrate for thymidylate synthase (TS) and DNA synthesis and repair. Although dUTP is a normal intermediate in DNA synthesis, its accumulation and misincorporation into DNA as uracil is lethal. Importantly, uracil misincorporation represents an important mechanism of cytotoxicity induced by the TS-targeted class of chemotherapeutic agents including 5-FU. A growing body of evidence suggests that dUTPase is an important mediator of response to TS-targeted agents. In this article, we present further evidence showing that elevated expression of dUTPase can protect breast cancer cells from the expansion of the intracellular uracil pool, translating to reduced growth inhibition following treatment with 5-FU. We therefore report the implementation of in silico drug development techniques to identify and develop small-molecule inhibitors of dUTPase. As 5-FU and the oral 5-FU prodrug capecitabine remain central agents in the treatment of a variety of malignancies, the clinical utility of a small-molecule inhibitor to dUTPase represents a viable strategy to improve the clinical efficacy of these mainstay chemotherapeutic agents.

Plastin Polymorphisms Predict Gender- and Stage-Specific Colon Cancer Recurrence after Adjuvant Chemotherapy

Tumor recurrence after curative resection remains a major problem in patients with locally advanced colorectal cancer treated with adjuvant chemotherapy. Genetic single-nucleotide polymorphisms (SNP) may serve as useful molecular markers to predict clinical outcomes in these patients and identify targets for future drug development. Recent in vitro and in vivo studies have demonstrated that the plastin genes PLS3 and LCP1 are overexpressed in colon cancer cells and play an important role in tumor cell invasion, adhesion, and migration. Hence, we hypothesized that functional genetic variations of plastin may have direct effects on the progression and prognosis of locally advanced colorectal cancer. We tested whether functional tagging polymorphisms of PLS3 and LCP1 predict time to tumor recurrence (TTR) in 732 patients (training set, 234; validation set, 498) with stage II/III colorectal cancer. The PLS3 rs11342 and LCP1 rs4941543 polymorphisms were associated with a significantly increased risk for recurrence in the training set. PLS3 rs6643869 showed a consistent association with TTR in the training and validation set, when stratified by gender and tumor location. Female patients with the PLS3 rs6643869 AA genotype had the shortest median TTR compared with those with any G allele in the training set [1.7 vs. 9.4 years; HR, 2.84; 95% confidence interval (CI), 1.32-6.1; P = 0.005] and validation set (3.3 vs. 13.7 years; HR, 2.07; 95% CI, 1.09-3.91; P = 0.021). Our findings suggest that several SNPs of the PLS3 and LCP1 genes could serve as gender- and/or stage-specific molecular predictors of tumor recurrence in stage II/III patients with colorectal cancer as well as potential therapeutic targets.

The Cyclin D1 (CCND1) A870G polymorphism predicts clinical outcome to lapatinib and capecitabine in HER2-positive metastatic breast cancer

BACKGROUND: Lapatinib plus capecitabine emerged as an efficacious therapy in metastatic breast cancer (mBC). We aimed to identify germline single-nucleotide polymorphisms (SNPs) in genes involved in capecitabine catabolism and human epidermal receptor signaling that were associated with clinical outcome to assist in selecting patients likely to benefit from this combination.

PATIENTS AND METHODS: DNA was extracted from 240 of 399 patients enrolled in EGF100151 clinical trial (NCT00078572; clinicaltrials.gov) and SNPs were successfully evaluated in 234 patients. The associations between SNPs and clinical outcome were analyzed using Fisher's exact test, Kaplan-Meier curves, log-rank tests, likelihood ratio test within logistic or Cox regression model, as appropriate.

RESULTS: There were significant interactions between CCND1 A870G and clinical outcome. Patients carrying the A-allele were more likely to benefit from lapatinib plus capecitabine versus capecitabine when compared with patients harboring G/G (P = 0.022, 0.024 and 0.04, respectively). In patients with the A-allele, the response rate (RR) was significantly higher with lapatinib plus capecitabine (35%) compared with capecitabine (11%; P = 0.001) but not between treatments in patients with G/G (RR = 24% and 32%, respectively; P = 0.85). Time to tumor progression (TTP) was longer in patients with the A-allele treated with lapatinib plus capecitabine compared with capecitabine (median TTP = 7.9 and 3.4 months; P < 0.001), but not in patients with G/G (median TTP = 6.1 and 6.6 months; P = 0.92).

CONCLUSION: Our findings suggest that CCND1A870G may be useful in predicting clinical outcome in HER2-positive mBC patients treated with lapatinib plus capecitabine.

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.

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.

Association of common gene variants in the WNT/β-catenin pathway with colon cancer recurrence

Wnt/β-catenin signaling has a central role in the development and progression of most colon cancers (CCs). Germline variants in Wnt/β-catenin pathway genes may result in altered gene function and/or activity, thereby causing inter-individual differences in relation to tumor recurrence capacity and chemoresistance. We investigated germline polymorphisms in a comprehensive panel of Wnt/β-catenin pathway genes to predict time to tumor recurrence (TTR) in patients with stage III and high-risk stage II CC. A total of 234 patients treated with 5-fluorouracil-based chemotherapy were included in this study. Whole-blood samples were analyzed for putative functional germline polymorphisms in SFRP3, SFRP4, DKK2, DKK3, Axin2, APC, TCF7L2, WNT5B, CXXC4, NOTCH2 and GLI1 genes by PCR-based restriction fragment-length polymorphism or direct DNA sequencing. Polymorphisms with statistical significance were validated in an independent study cohort. The minor allele of WNT5B rs2010851 T>G was significantly associated with a shorter TTR (10.7 vs 4.9 years; hazard ratio: 2.48; 95% CI, 0.96-6.38; P=0.04) in high-risk stage II CC patients. This result remained significant in multivariate Cox's regression analysis. This study shows that the WNT5B germline variant rs2010851 was significantly identified as a stage-dependent prognostic marker for CC patients after 5-fluorouracil-based adjuvant therapy.

Gender-specific profiling in SCN1A polymorphisms and time-to-recurrence in patients with stage II/III colorectal cancer treated with adjuvant 5-fluoruracil chemotherapy

This study was designed to analyze the gender-related association between SCN1A polymorphisms (voltage-gated sodium channels; α-subunit) and time-to-recurrence (TTR) in patients with colorectal cancer (CRC) treated with 5-fluoruracil (5-FU)-based adjuvant chemotherapy. We enrolled from a prospective database patients with stage II and III CRC treated with adjuvant 5-FU-based chemotherapy. Genotypes for SCN1A rs3812718 and rs229877 were determined by direct DNA sequencing. One hundred twenty-seven males and 107 females were included in the study. In the univariate and multivariate analysis, the shortest TTR was associated with female patients carrying the rs3812718-TT genotype (hazard ratio (HR): 2.26 (95% confidence interval (CI): 0.89, 5.70), P=0.039) but with male patients carrying the rs3812718-CC genotype (HR: 0.49 (95% CI: 0.18, 1.38), P=0.048). For rs229877 the CT genotype was associated with a trend for shorter TTR in both gender populations. The study validated gender-dependent association between genomic SCN1A rs3812718 polymorphism and TTR in CRC patients treated with adjuvant 5-FU-based chemotherapy. This study confirms that voltage-gated Na+ channels may be a potential therapeutic target and a useful predictive biomarker before 5-FU infusion.

The cyclin D1 (CCND1) rs9344 G>A polymorphism predicts clinical outcome in colon cancer patients treated with adjuvant 5-FU-based chemotherapy

Recent evidence indicates a potential prognostic and predictive value for germline polymorphisms in genes involved in cell cycle control. We investigated the effect of cyclin D1 (CCND1) rs9344 G>A in stage II/III colon cancer patients and validated the findings in an independent study cohort. For evaluation and validation set, a total of 264 and 234 patients were included. Patients treated with 5-fluorouracil-based chemotherapy, carrying the CCND1 rs9344 A/A genotype had significantly decreased time-to-tumor recurrence (TTR) in univariate analysis and multivariate analysis (hazard ratio (HR) 2.47; 95% confidence interval (CI) 1.16-5.29; P=0.019). There was no significant association between CCND1 rs9344 G>A and TTR in patients with curative surgery alone. In the validation set, the A allele of CCND1 rs9344 G>A remained significantly associated with decreased TTR in univariate and multivariate analyses (HR 1.94; 95% CI 1.05-3.58; P=0.035). CCND1 rs9344 G>A may be a predictive and/or prognostic biomarker in stage II/III colon cancer patients, however, prospective trials are warranted to confirm our findings.

Pharmacogenomics and metastatic colorectal cancer:current knowledge and perspectives

The pharmacogenomics field is crucial for optimizing the selection of which chemotherapy regimen to use according to the patient's genomic profile. Indeed, the individual's inherited genome accounts for a large proportion of the variation in his or her response to chemotherapeutic agents both in terms of efficiency and toxicity. Patients with metastatic disease are more likely to receive different lines of chemotherapy with variable efficacy and experience some related complications. It is therefore critical to tailor the best therapeutic arsenal to improve the efficacy and avoid as much as possible related complications that are susceptible to interrupt the treatment. The pharmacogenomics approach investigates for each drug the implicated metabolic pathway and the potential personal variations in gene function. The aim of this review is to present a clear overview of the most accurate polymorphisms that have been identified as related to drug response in patients with mCRC.

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 patient's 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.

A review of excision repair cross-complementation group 1 in colorectal cancer

Oxaliplatin-based chemotherapy is the standard of care in patients with high-risk stage II and stage III colorectal cancer as well as in patients with advanced disease. Unfortunately, a large proportion of patients offered oxaliplatin fail to benefit from it. In the era of personalized treatment, there are strong efforts to identify biomarkers that will predict efficacy to oxaliplatin-based treatments. Excision repair cross-complementation group 1 (ERCC1) is a key element in the nucleotide excision repair (NER) pathway, which is responsible for repairing DNA adducts induced by platinum compounds. ERCC1 has recently been shown to be closely associated with outcome in patients with non-small-cell lung cancer (NSCLC): both high ERCC1 protein and gene expression are associated with resistance to cisplatin-based chemotherapy and better outcome without treatment. Therefore, ERCC1 has the potential to be used as a strong candidate biomarker, both predictive and prognostic, for colorectal cancer. This review will focus on the preclinical and clinical evidences supporting ERCC1 as a major molecule in oxaliplatin resistance. In addition, the important technologies used to assess ERCC1 gene and protein expression will be highlighted.