Tumor Epithelial Cell Matrix Metalloproteinase 9 (MMP-9) is a Prognostic Marker in Colorectal Cancer

Presented at American Association Cancer Research in 2008 Zuzga D.S., Gibbons A.V., Li P., Lubbe W.J., Chervoneva I., Pitari G.M. “Tumor epithelial cell MMP-9 is a prognostic marker in colorectal cancer”. In: American Association for Cancer Research Special Conference, Molecular Diagnostics in Cancer Therapeutic Development: Proceedings; 2008 Sept 22-25; Philadelphia, PA. Abstract A40. Colorectal cancer is the second leading cause of cancer-related mortality indeveloped nations. Mortality from colon cancer largely reflects metastasis, thespread of the disease to distant sites. Early diagnosis of pre-metastatic diseaseand accurate stratification of patients with metastasis is pivotal to decreasemortality rates from colon cancer by effectively administering surgery alone orwith chemotherapy. However, specific pathological markers of colorectal cancermetastasis have not emerged. Matrix metalloproteinase 9 (MMP-9) is a keyregulator of metastasis and a therapeutic target in colon cancer. Here, MMP-9overexpression in pure tumor epithelial, but nor stromal, cell populations frompatients was associated with metastatic colorectal cancer progression as definedby RT-PCR and confirmed by immunostaining. Thus, tumors with increasedMMP-9 expression compared to matched normal adjacent tissues alwaysexhibited metastatic dissemination. In particular, MMP-9 overexpression in tumorepithelial cells, compared to normal epithelial cells, specifically predicted lymphnode involvement. Importantly, patients with relative increase of MMP-9 levels intumor epithelial cells were characterized by more advanced disease stages, withsignificantly higher proportion of regional lymph nodes harboring metastasis,compared to patients with a relative decrease in MMP-9 expression. Together,these observations suggest tumor epithelial cell MMP-9 is a novel prognosticmarker that may be exploited for more efficient disease stage stratification andtherapeutic regimen selection in patients with colorectal cancer

Reversibility of Apixaban Anticoagulation with a Four-Factor Prothrombin Complex Concentrate in Healthy Volunteers.

It was hypothesized that the four-factor prothrombin complex concentrate (4F-PCC) Kcentra 25 unit/kg would reverse impairment of thrombin generation in healthy volunteers dosed with apixaban to steady state. In this randomized, two-period crossover, assessor-blinded trial, 12 healthy subjects received 5 mg apixaban every 12 h. Three h after the fifth dose, four-factor prothrombin complex concentrate (4F-PCC) 25 unit/kg or saline were infused. Serial blood samples were assessed for thrombin generation using PPP-reagent and PPP-reagent low, anti-Xa, PT, and PTT assays. Geometric mean ratio was calculated at 30 min postinfusion, and at 24, 48, and 72 h. Peak thrombin generation was 76% higher at 30 min postinfusion with 4F-PCC (p = 0.025). The difference declined to 24% at 24 h and resolved by 48 h. Other thrombin generation parameters were also partially normalized. There was no difference between 4F-PCC and saline in anti-Xa assessment at 30 min or later time points

The effects of CEP-37440, an inhibitor of focal adhesion kinase, in vitro and in vivo on inflammatory breast cancer cells.

BACKGROUND: Inflammatory breast cancer (IBC) is an aggressive type of advanced breast cancer with a poor prognosis. We recently found that focal adhesion kinase 1 (FAK1) is upregulated and phosphorylated (active) in IBC. In this study, we investigated the effect of CEP-37440, a dual inhibitor of FAK1 and anaplastic lymphoma kinase (ALK), using human IBC cell lines and preclinical models of IBC. METHODS: Cell proliferation assays were performed in the presence of several concentrations of CEP-37440 using IBC and triple-negative breast cancer non-IBC cell lines. In vitro, we studied the expression of total FAK1, phospho-FAK1 (Tyr 397), total ALK and phospho-ALK (Tyr 1604). In vivo, we tested CEP-37440 using FC-IBC02, SUM149, and SUM190 IBC xenograft mouse models. RESULTS: CEP-37440 at low concentration decreased the proliferation of the IBC cell lines FC-IBC02, SUM190, and KPL4, while not affecting the proliferation of normal breast epithelial cells. At higher concentration, CEP-37440 was also able to inhibit the proliferation of the IBC cell line MDA-IBC03 and the triple-negative non-IBC cell lines MDA-MB-231 and MDA-MB-468; the IBC cell line SUM149 showed a slight response to the drug. CEP-37440 decreased the cell proliferation of FC-IBC02, SUM190, and KPL4 by blocking the autophosphorylation kinase activity of FAK1 (Tyr 397). None of the cells evaluated expressed ALK. In vivo, after 7 weeks of CEP-37440 treatment, the SUM190, FC-IBC02, and SUM149 breast tumor xenografts were smaller in mice treated with 55 mg/kg bid CEP-37440 compared to the controls; the tumor growth inhibition (TGI) was 79.7 %, 33 %, and 23 %, respectively. None of the FC-IBC02 breast xenografts mice treated with CEP-37440 developed brain metastasis while 20 % of the mice in the control group developed brain metastasis. Expression array analyses in FC-IBC02 cells showed that CEP-37440 affects the expression of genes related to apoptosis, interferon signaling, and cytokines. CONCLUSIONS: CEP-37440 is effective against some IBC cells that express phospho-FAK1 (Tyr 397), and its antiproliferative activity is related to its ability to decrease phospho-FAK1. Our results suggest that combinational therapies could be more effective than using CEP-37440 as a single agent

Proteomic analysis of nipple aspirate fluid to detect biologic markers of breast cancer.

The early detection of breast cancer is the best means to minimise disease-related mortality. Current screening techniques have limited sensitivity and specificity. Breast nipple aspirate fluid can be obtained noninvasively and contains proteins secreted from ductal and lobular epithelia. Nipple aspirate fluid proteins are breast specific and generally more concentrated than corresponding blood levels. Proteomic analysis of 1 microl of diluted nipple aspirate fluid over a 5-40 kDa range from 20 subjects with breast cancer and 13 with nondiseased breasts identified five differentially expressed proteins. The most sensitive and specific proteins were 6500 and 15 940 Da, found in 75-84% of samples from women with cancer but in only 0-9% of samples from normal women. These findings suggest that (1) differential expression of nipple aspirate fluid proteins exists between women with normal and diseased breasts, and (2) analysis of these proteins may predict the presence of breast cancer

SOX10 requirement for melanoma tumor growth is due, in part, to immune-mediated effects

Developmental factors may regulate the expression of immune modulatory proteins in cancer, linking embryonic development and cancer cell immune evasion. This is particularly relevant in melanoma because immune checkpoint inhibitors are commonly used in the clinic. SRY-box transcription factor 10 (SOX10) mediates neural crest development and is required for melanoma cell growth. In this study, we investigate immune-related targets of SOX10 and observe positive regulation of herpesvirus entry mediator (HVEM) and carcinoembryonic-antigen cell-adhesion molecule 1 (CEACAM1). Sox10 knockout reduces tumor growth in vivo, and this effect is exacerbated in immune-competent models. Modulation of CEACAM1 expression but not HVEM elicits modest effects on tumor growth. Importantly, Sox10 knockout effects on tumor growth are dependent, in part, on CD8+ T cells. Extending this analysis to samples from patients with cutaneous melanoma, we observe a negative correlation with SOX10 and immune-related pathways. These data demonstrate a role for SOX10 in regulating immune checkpoint protein expression and anti-tumor immunity in melanoma

Spatial Metrics of Interaction between CD163-Positive Macrophages and Cancer Cells and Progression-Free Survival in Chemo-Treated Breast Cancer

Tumor-associated macrophages (TAMs) promote progression of breast cancer and other solid malignancies via immunosuppressive, pro-angiogenic and pro-metastatic effects. Tumor-promoting TAMs tend to express M2-like macrophage markers, including CD163. Histopathological assessments suggest that the density of CD163-positive TAMs within the tumor microenvironment is associated with reduced efficacy of chemotherapy and unfavorable prognosis. However, previous analyses have required research-oriented pathologists to visually enumerate CD163+ TAMs, which is both laborious and subjective and hampers clinical implementation. Objective, operator-independent image analysis methods to quantify TAM-associated information are needed. In addition, since M2-like TAMs exert local effects on cancer cells through direct juxtacrine cell-to-cell interactions, paracrine signaling, and metabolic factors, we hypothesized that spatial metrics of adjacency of M2-like TAMs to breast cancer cells will have further information value. Immunofluorescence histo-cytometry of CD163+ TAMs was performed retrospectively on tumor microarrays of 443 cases of invasive breast cancer from patients who subsequently received adjuvant chemotherapy. An objective and automated algorithm was developed to phenotype CD163+ TAMs and calculate their density within the tumor stroma and derive several spatial metrics of interaction with cancer cells. Shorter progression-free survival was associated with a high density of CD163+ TAMs, shorter median cancer-to-CD163+ nearest neighbor distance, and a high number of either directly adjacent CD163+ TAMs (within juxtacrine proximity \u3c12 µm to cancer cells) or communicating CD163+ TAMs (within paracrine communication distance \u3c250 µm to cancer cells) after multivariable adjustment for clinical and pathological risk factors and correction for optimistic bias due to dichotomization

Targeting SOX10-deficient cells to reduce the dormant-invasive phenotype state in melanoma

Cellular plasticity contributes to intra-tumoral heterogeneity and phenotype switching, which enable adaptation to metastatic microenvironments and resistance to therapies. Mechanisms underlying tumor cell plasticity remain poorly understood. SOX10, a neural crest lineage transcription factor, is heterogeneously expressed in melanomas. Loss of SOX10 reduces proliferation, leads to invasive properties, including the expression of mesenchymal genes and extracellular matrix, and promotes tolerance to BRAF and/or MEK inhibitors. We identify the class of cellular inhibitor of apoptosis protein-1/2 (cIAP1/2) inhibitors as inducing cell death selectively in SOX10-deficient cells. Targeted therapy selects for SOX10 knockout cells underscoring their drug tolerant properties. Combining cIAP1/2 inhibitor with BRAF/MEK inhibitors delays the onset of acquired resistance in melanomas in vivo. These data suggest that SOX10 mediates phenotypic switching in cutaneous melanoma to produce a targeted inhibitor tolerant state that is likely a prelude to the acquisition of resistance. Furthermore, we provide a therapeutic strategy to selectively eliminate SOX10-deficient cells

Fine-Mapping, Gene Expression and Splicing Analysis of the Disease Associated LRRK2 Locus

Association studies have identified several signals at the LRRK2 locus for Parkinson's disease (PD), Crohn's disease (CD) and leprosy. However, little is known about the molecular mechanisms mediating these effects. To further characterize this locus, we fine-mapped the risk association in 5,802 PD and 5,556 controls using a dense genotyping array (ImmunoChip). Using samples from 134 post-mortem control adult human brains (UK Human Brain Expression Consortium), where up to ten brain regions were available per individual, we studied the regional variation, splicing and regulation of LRRK2. We found convincing evidence for a common variant PD association located outside of the LRRK2 protein coding region (rs117762348, A>G, P = 2.56×10(-8), case/control MAF 0.083/0.074, odds ratio 0.86 for the minor allele with 95% confidence interval [0.80-0.91]). We show that mRNA expression levels are highest in cortical regions and lowest in cerebellum. We find an exon quantitative trait locus (QTL) in brain samples that localizes to exons 32-33 and investigate the molecular basis of this eQTL using RNA-Seq data in n = 8 brain samples. The genotype underlying this eQTL is in strong linkage disequilibrium with the CD associated non-synonymous SNP rs3761863 (M2397T). We found two additional QTLs in liver and monocyte samples but none of these explained the common variant PD association at rs117762348. Our results characterize the LRRK2 locus, and highlight the importance and difficulties of fine-mapping and integration of multiple datasets to delineate pathogenic variants and thus develop an understanding of disease mechanisms