Proteomic analysis of colorectal cancer metastasis:stathmin-1 revealed as a player in cancer cell migration and prognostic marker

Metastasis accounts largely for the high mortality rate of colorectal cancer (CRC) patients. In this study, we performed comparative proteome analysis of primary CRC cell lines HCT-116 and its metastatic derivative E1 using 2-D DIGE. We identified 74 differentially expressed proteins, many of which function in transcription, translation, angiogenesis signal transduction, or cytoskeletal remodeling pathways, which are indispensable cellular processes involved in the metastatic cascade. Among these proteins, stathmin-1 (STMN1) was found to be highly up-regulated in E1 as compared to HCT-116 and was thus selected for further functional studies. Our results showed that perturbations in STMN1 levels resulted in significant changes in cell migration, invasion, adhesion, and colony formation. We further showed that the differential expression of STMN1 correlated with the cells' metastatic potential in other paradigms of CRC models. Using immunohistochemistry, we also showed that STMN1 was highly expressed in colorectal primary tumors and metastatic tissues as compared to the adjacent normal colorectal tissues. Furthermore, we also showed via tissue microarray analyses of 324 CRC tissues and Kaplan-Meier survival plot that CRC patients with higher expression of STMN1 have poorer prognosis.

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.

Coexpressing shRNA with fluorescence tags for quantification of cell migration studies

Understanding migration of cells has many implications in human physiology; some examples include developmental biology, healing, immune responses and tissue remodeling. On the other hand, invasive migration by tumor cells is pathological and is a major cause of mortality amongst cancer sufferers. Cell migration assays have been widely used to quantify potentially metastatic genes. In recent years, the use of RNAi has significantly increased the tools available in cell migration research due to its specific gene targeting for knockdown. The inability to ensure 100% transfection/transduction efficiency reduces the sensitivity of cell migration assays because cells not successfully transfected/transduced with the RNAi are also included in the calculations. This study introduces a different experimental setup mathematically expressed in our named normalized relative infected cell count (N-RICC) that analyses cell migration assays by co-expressing retrovirally transduced shRNA with fluorescence tags from a single vector. Vectors transduced into cells are visible under fluorescence, thus alleviating the problems involved with transduction efficiency by individually identifying cells with targeted genes. Designed shRNAs were targeted against a list of potentially metastatic genes in a highly migratory breast cancer cell line model, MDA-MB-231. We have successfully applied N-RICC analysis to show greater sensitivity of integrin alpha5 (ITGA5) and Ras homologue A (RhoA) in cell metastasis over conventional methods in scratch-wound assays and migration chambers assays.

Overexpression of neurone glial-related cell adhesion molecule is an independent predictor of poor prognosis in advanced colorectal cancer

A downstream target of the Wnt pathway, neurone glial-related cell adhesion molecule (Nr-CAM) has recently been implicated in human cancer development. However, its role in colorectal cancer (CRC) pathobiology and clinical relevance remains unknown. In this study, we examined the clinical significance of Nr-CAM protein expression in a retrospective series of 428 CRCs using immunohistochemistry and tissue microarrays. Cox proportional hazards regression was used to calculate hazard ratios (HR) of mortality according to various clinicopathological features and molecular markers. All CRC samples were immunoreactive for Nr-CAM protein expression, compared to 10 / 245 (4%) matched normal tissue (P <0.0001). Of 428 CRC samples, 97 (23%) showed Nr-CAM overexpression, which was significantly associated with nodal (P = 0.012) and distant (P = 0.039) metastasis, but not with extent of local invasion or tumor size. Additionally, Nr-CAM overexpression was associated with vascular invasion (P = 0.0029), p53 expression (P = 0.036), and peritoneal metastasis at diagnosis (P = 0.013). In a multivariate model adjusted for other clinicopathological predictors of survival, Nr-CAM overexpression correlated with a significant increase in disease-specific (HR 1.66; 95% confidence interval 1.11-2.47; P = 0.014) and overall mortality (HR 1.57; 95% confidence interval 1.07-2.30; P = 0.023) in advanced but not early stage disease. Notably, 5-fluorouracil-based chemotherapy conferred significant survival benefit to patients with tumors negative for Nr-CAM overexpression but not to those with Nr-CAM overexpressed tumors. In conclusion, Nr-CAM protein expression is upregulated in CRC tissues. Nr-CAM overexpression is an independent marker of poor prognosis among advanced CRC patients, and is a possible predictive marker for non-beneficence to 5-fluorouracil- based chemotherapy.

DEAD-box helicase DP103 defines metastatic potential of human breast cancers

Despite advancement in breast cancer treatment, 30% of patients with early breast cancers experience relapse with distant metastasis. It is a challenge to identify patients at risk for relapse; therefore, the identification of markers and therapeutic targets for metastatic breast cancers is imperative. Here, we identified DP103 as a biomarker and metastasis-driving oncogene in human breast cancers and determined that DP103 elevates matrix metallopeptidase 9 (MMP9) levels, which are associated with metastasis and invasion through activation of NF-κB. In turn, NF-κB signaling positively activated DP103 expression. Furthermore, DP103 enhanced TGF-β-activated kinase-1 (TAK1) phosphorylation of NF-κB-activating IκB kinase 2 (IKK2), leading to increased NF-κB activity. Reduction of DP103 expression in invasive breast cancer cells reduced phosphorylation of IKK2, abrogated NF-κB-mediated MMP9 expression, and impeded metastasis in a murine xenograft model. In breast cancer patient tissues, elevated levels of DP103 correlated with enhanced MMP9, reduced overall survival, and reduced survival after relapse. Together, these data indicate that a positive DP103/NF-κB feedback loop promotes constitutive NF-κB activation in invasive breast cancers and activation of this pathway is linked to cancer progression and the acquisition of chemotherapy resistance. Furthermore, our results suggest that DP103 has potential as a therapeutic target for breast cancer treatment.

Randomized Phase II trial of nintedanib, afatinib and sequential combination in castration-resistant prostate cancer

AIMS: The aim of this article was to evaluate afatinib (BIBW 2992), an ErbB family blocker, and nintedanib (BIBF 1120), a triple angiokinase inhibitor, in castration-resistant prostate cancer patients.

PATIENTS & METHODS: Patients were randomized to receive nintedanib (250 mg twice daily), afatinib (40 mg once daily [q.d.]), or alternating sequential 7-day nintedanib (250 mg twice daily) and afatinib (70 mg q.d. [Combi70]), which was reduced to 40 mg q.d. (Combi40) due to adverse events. The primary end point was progression-free rate at 12 weeks.

RESULTS: Of the 85 patients treated 46, 20, 16 and three received nintedanib, afatinib, Combi40 and Combi70, respectively. At 12 weeks, the progression-free rate was 26% (seven out of 27 patients) for nintedanib, and 0% for afatinib and Combi40 groups. Two patients had a ≥50% decline in PSA (nintedanib and the Combi40 groups). The most common drug-related adverse events were diarrhea, nausea, vomiting and lethargy.

CONCLUSION: Nintedanib and/or afatinib demonstrated limited anti-tumor activity in unselected advanced castration-resistant prostate cancer patients.

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.

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.

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.

Gender-specific genomic profiling in metastatic colorectal cancer patients treated with 5-fluorouracil and oxaliplatin

AIMS: Survival and response rates in metastatic colorectal cancer remain poor, despite advances in drug development. There is increasing evidence to suggest that gender-specific differences may contribute to poor clinical outcome. We tested the hypothesis that genomic profiling of metastatic colorectal cancer is dependent on gender.

MATERIALS & METHODS: A total of 152 patients with metastatic colorectal cancer who were treated with oxaliplatin and continuous infusion 5-fluorouracil were genotyped for 21 polymorphisms in 13 cancer-related genes by PCR. Classification and regression tree analysis tested for gender-related association of polymorphisms with overall survival, progression-free survival and tumor response.

RESULTS: Classification and regression tree analysis of all polymorphisms, age and race resulted in gender-specific predictors of overall survival, progression-free survival and tumor response. Polymorphisms in the following genes were associated with gender-specific clinical outcome: estrogen receptor β, EGF receptor, xeroderma pigmentosum group D, voltage-gated sodium channel and phospholipase A2.

CONCLUSION: Genetic profiling to predict the clinical outcome of patients with metastatic colorectal cancer may depend on gender.

DNA-Repair Defects and Olaparib in Metastatic Prostate Cancer

BACKGROUND: Prostate cancer is a heterogeneous disease, but current treatments are not based on molecular stratification. We hypothesized that metastatic, castration-resistant prostate cancers with DNA-repair defects would respond to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibition with olaparib.

METHODS: We conducted a phase 2 trial in which patients with metastatic, castration-resistant prostate cancer were treated with olaparib tablets at a dose of 400 mg twice a day. The primary end point was the response rate, defined either as an objective response according to Response Evaluation Criteria in Solid Tumors, version 1.1, or as a reduction of at least 50% in the prostate-specific antigen level or a confirmed reduction in the circulating tumor-cell count from 5 or more cells per 7.5 ml of blood to less than 5 cells per 7.5 ml. Targeted next-generation sequencing, exome and transcriptome analysis, and digital polymerase-chain-reaction testing were performed on samples from mandated tumor biopsies.

RESULTS: Overall, 50 patients were enrolled; all had received prior treatment with docetaxel, 49 (98%) had received abiraterone or enzalutamide, and 29 (58%) had received cabazitaxel. Sixteen of 49 patients who could be evaluated had a response (33%; 95% confidence interval, 20 to 48), with 12 patients receiving the study treatment for more than 6 months. Next-generation sequencing identified homozygous deletions, deleterious mutations, or both in DNA-repair genes--including BRCA1/2, ATM, Fanconi's anemia genes, and CHEK2--in 16 of 49 patients who could be evaluated (33%). Of these 16 patients, 14 (88%) had a response to olaparib, including all 7 patients with BRCA2 loss (4 with biallelic somatic loss, and 3 with germline mutations) and 4 of 5 with ATM aberrations. The specificity of the biomarker suite was 94%. Anemia (in 10 of the 50 patients [20%]) and fatigue (in 6 [12%]) were the most common grade 3 or 4 adverse events, findings that are consistent with previous studies of olaparib.

CONCLUSIONS: Treatment with the PARP inhibitor olaparib in patients whose prostate cancers were no longer responding to standard treatments and who had defects in DNA-repair genes led to a high response rate. (Funded by Cancer Research UK and others; ClinicalTrials.gov number, NCT01682772; Cancer Research UK number, CRUK/11/029.).

Metastasis-inducing DNA Regulates the Expression of the Osteopontin Gene by Binding the Transcription Factor Tcf-4

Small 1,000-bp fragments of genomic DNA obtained from human malignant breast cancer cell lines when transfected into a benign rat mammary cell line enhance transcription of the osteopontin gene and thereby cause the cells to metastasize in syngeneic rats. To identify the molecular events underlying this process, transient cotransfections of an osteopontin promoter-reporter construct and fragments of one metastasis-inducing DNA (Met-DNA) have identified the active components in the Met-DNA as the binding sites for the T-cell factor (Tcf) family of transcription factors. Incubation of cell extracts with active DNA fragments containing the sequence CAAAG caused retardation of their mobilities on polyacrylamide gels, and Western blotting identified Tcf-4, beta-catenin, and E-cadherin in the relevant DNA complexes in vitro. Transfection of an expression vector for Tcf-4 inhibited the stimulated activity of the osteopontin promoter-reporter construct caused by transiently transfected active fragments of Met-DNA or permanently transfected Met-DNA. This stimulated activity of the osteopontin promoter-reporter construct is accompanied by an increase in endogenous osteopontin mRNA but not in fos or actin mRNAs in the transfected cells. Permanent transfection of the benign rat mammary cell line with a 20-bp fragment from the Met-DNA containing the Tcf recognition sequence CAAAG caused an enhanced permanent production of endogenous osteopontin protein in vitro and induced the cells to metastasize in syngeneic rats in vivo. The corresponding fragment without the CAAAG sequence was without either effect. Therefore, the regulatory effect of the C9-Met-DNA is exerted, at least in part, by a CAAAG sequence that can sequester the endogenous inhibitory Tcf-4 and thereby promote transcription of osteopontin, the direct effector of metastasis in this system.