Nicotine Promotes Tumor Growth and Metastasis in Mouse Models of Lung Cancer

Nicotine is the major addictive component of tobacco smoke. Although nicotine is generally thought to have limited ability to initiate cancer, it can induce cell proliferation and angiogenesis in a variety of systems. These properties might enable nicotine to facilitate the growth of tumors already initiated. Here we show that nicotine significantly promotes the progression and metastasis of tumors in mouse models of lung cancer. This effect was observed when nicotine was administered through intraperitoneal injections, or through over-the-counter transdermal patches.In the present study, Line1 mouse adenocarcinoma cells were implanted subcutaneously into syngenic BALB/c mice. Nicotine administration either by intraperitoneal (i.p.) injection or transdermal patches caused a remarkable increase in the size of implanted Line1 tumors. Once the tumors were surgically removed, nicotine treated mice had a markedly higher tumor recurrence (59.7%) as compared to the vehicle treated mice (19.5%). Nicotine also increased metastasis of dorsally implanted Line1 tumors to the lungs by 9 folds. These studies on transplanted tumors were extended to a mouse model where the tumors were induced by the tobacco carcinogen, NNK. Lung tumors were initiated in A/J mice by i.p. injection of NNK; administration of 1 mg/kg nicotine three times a week led to an increase in the size and the number of tumors formed in the lungs. In addition, nicotine significantly reduced the expression of epithelial markers, E-Cadherin and beta-Catenin as well as the tight junction protein ZO-1; these tumors also showed an increased expression of the alpha(7) nAChR subunit. We believe that exposure to nicotine either by tobacco smoke or nicotine supplements might facilitate increased tumor growth and metastasis.Our earlier results indicated that nicotine could induce invasion and epithelial-mesenchymal transition (EMT) in cultured lung, breast and pancreatic cancer cells. This study demonstrates for the first time that administration of nicotine either by i.p. injection or through over-the-counter dermal patches can promote tumor growth and metastasis in immunocompetent mice. These results suggest that while nicotine has only limited capacity to initiate tumor formation, it can facilitate the progression and metastasis of tumors pre-initiated by tobacco carcinogens

Hepatocyte Growth Factor from a Clinical Perspective: A Pancreatic Cancer Challenge

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States and incidence rates are rising. Both detection and treatment options for pancreatic cancer are limited, providing a less than 5% five-year survival advantage. The need for new biomarkers for early detection and treatment of pancreatic cancer demands the efficient translation of bench knowledge to provide clinical benefit. One source of therapeutic resistance is the pancreatic tumor microenvironment, which is characterized by desmoplasia and hypoxia making it less conducive to current therapies. A major factor regulating desmoplasia and subsequently promoting chemoresistance in pancreatic cancer is hepatocyte growth factor (HGF), the sole ligand for c-MET (mesenchymal-epithelial transition), an epithelial tyrosine kinase receptor. Binding of HGF to c-MET leads to receptor dimerization and autophosphorylation resulting in the activation of multiple cellular processes that support cancer progression. Inhibiting activation of c-MET in cancer cells, in combination with other approaches for reducing desmoplasia in the tumor microenvironment, might significantly improve the success of chemotherapy. Therefore, HGF makes a potent novel target for developing therapeutic strategies in combination with existing drugs for treating pancreatic adenocarcinoma. This review provides a comprehensive analysis of HGF and its promising potential as a chemotherapeutic target for pancreatic cancer

Mammalian lysine histone demethylase KDM2A regulates E2F1-mediated gene transcription in breast cancer cells.

It is established that histone modifications like acetylation, methylation, phosphorylation and ubiquitination affect chromatin structure and modulate gene expression. Lysine methylation/demethylation on Histone H3 and H4 is known to affect transcription and is mediated by histone methyl transferases and histone demethylases. KDM2A/JHDM1A/FBXL11 is a JmjC-containing histone demethylase that targets mono- and dimethylated Lys36 residues of Histone H3; its function in breast cancer is not fully understood. Here we show that KDM2A is strongly expressed in myoepithelial cells (MEPC) in breast cancer tissues by immunohistochemistry. Ductal cells from ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC) show positive staining for KDM2A, the expression decreases with disease progression to metastasis. Since breast MEPCs have tumor-suppressive and anti-angiogenic properties, we hypothesized that KDM2A could be contributing to some of these functions. Silencing KDM2A with small interfering RNAs demonstrated increased invasion and migration of breast cancer cells by suppressing a subset of matrix metalloproteinases (MMP-2, -9, -14 and -15), as seen by real-time PCR. HUVEC cells showed increased angiogenic tubule formation ability in the absence of KDM2A, with a concomitant increase in the expression of VEGF receptors, FLT-1 and KDR. KDM2A physically bound to both Rb and E2F1 in a cell cycle dependent manner and repressed E2F1 transcriptional activity. Chromatin immunoprecipitation (ChIP) assays revealed that KDM2A associates with E2F1-regulated proliferative promoters CDC25A and TS in early G-phase and dissociates in S-phase. Further, KDM2A could also be detected on MMP9, 14 and 15 promoters, as well as promoters of FLT1 and KDR. KDM2A could suppress E2F1-mediated induction of these promoters in transient transfection experiments. These results suggest a regulatory role for KDM2A in breast cancer cell invasion and migration, through the regulation of E2F1 function

Influencing factors on the NMP-22 urine assay: an experimental model

Abstract Background The commercial NMP-22 urine assays for bladder cancer (BCa) detect nuclear mitotic apparatus protein 1 (NUMA1) using monoclonal antibodies. It remains unclear whether these assays are monitoring a tumor antigen or some other phenomenon associated with the disease state. In this study, we investigated the influence of urinary cellular and protein concentration, and hematuria on the performance of the NMP-22 tests in an experimental model. Methods Pooled urine from healthy subjects were spiked with varying concentrations of benign (UROtsa) cells, cancer cells (RT4, T24, KU-7 and UM-UC-14), whole blood or serum, prior to analysis with both NMP22® Bladder Cancer ELISA test and the NMP22® BladderChek® point-of-care test. Results Urines from control subjects were negative for NMP-22. The addition of whole blood at 50ul/10 ml, but not serum, resulted in a false-positive result. Furthermore, the addition of a high concentration of benign urothelial cells (106) or the cell lysate from these cells (306 μg protein) resulted in a false-positive result. High concentrations of pooled-cancer cells (106) or cell lysate (30.6 μg and above) resulted in a positive NMP-22 assay. Concordance between the NMP-22 ELISA assay and the NMP-22 point of care assay was >90%. Conclusions Rather than detecting a specific tumor antigen, urinary NMP-22 assays may be measuring the cellularity or amount of cell turnover that may be introduced into the urine by a variety of conditions, including surface shedding from bladder tumors. The absence of significant urinary cellularity in some cases due to lesion characteristics or the timing of sampling may result in false-negative NMP-2 assays.</p

KDM2A interacts with Rb and E2F1 in a cell cycle specific manner.

<p>(A) GST pull down assay showing KDM2A binding to Rb and E2F1 <i>in vitro</i>. ³⁵S-lysate lane has 1/10^th protein loaded. (B) MCF-7 cells were serum starved for 48 hr and serum stimulated for 2 hr, 4 hr, 6 hr, 8 hr and 18 hr. Western blotting showing protein expression of KDM2A, Rb, E2F1 and actin. There is no significant change in the expression of KDM2A and E2F1 at different time points when normalized to actin levels. Rb shows increasing hyperphosphorylation from 6 hr to 18 hr of serum stimulation. (C) KDM2A interacts with Rb and E2F1 <i>in vivo</i> in MCF-7 cells as seen by immunoprecipitation-western blot experiment. Interaction of KDM2A with E2F1 decreases at 8 hr of serum stimulation. (D) Chromatin Immunoprecipitation (ChIP) assays showing KDM2A occupancy on E2F1-regulated proliferative promoters, CDC25A and TS. KDM2A occupies the promoters at all time points from 0 hr to 6 hr, reduction is seen at 8 hr and complete absence of KDM2A from the promoters is observed at 18 hr of serum stimulation. (E) KDM2A significantly represses E2F1-mediated E2.Luc transcription in a dose-dependent manner in Renilla luciferase assay.</p

KDM2A represses transcriptional activity of MMPs and VEGF receptors by inhibiting E2F1-mediated transcription.

<p>(A) ChIP assay demonstrates occupancy of KDM2A on MMP9, -14, -15, KDR and FLT-1 similar to E2F1. (B, C, D, E) Transient transfection experiments in MCF-7 cells showed that E2F1 induces MMP2 (B), MMP-9 (C), MMP-14 (D) and MMP-15 (E) promoters, and this was repressed by co-transfection of KDM2A or Rb large pocket. (F, G) Transient transfection experiments in MDA-MB-231 cells demonstrate that KDM2A represses the transcriptional activity of E2F1 on KDR (F) and FLT-1 (G) promoters.</p

A model depicting KDM2A function in a mammalian cell.

<p>It can be implied that KDM2A regulates Rb-E2F1 function in the progression of the cell cycle. In a quiescent state, KDM2A represses E2F1 functions on various promoters. Addition of VEGF or serum dissociates KDM2A from these promoters facilitating various cellular processes by transcriptional activation leading to enhanced angiogenesis, invasion and migration of cells.</p

KDM2A suppresses invasion and migration of breast cancer cells.

<p>(A) Western blot analysis showing decreased KDM2A levels in T47D and MCF7 cells with two different siRNAs to KDM2A (Ambion and Santa Cruz) compared to control siRNA. (B) Silencing KDM2A by KDM2A siRNA 1 increased invasion in MCF7 cells by 145±23% (p<0.05) and KDM2A siRNA 2 increased invasion by 118±21% (p<0.05) when compared to control siRNA. (C) In T47D cells, KDM2A siRNA 1 enhanced invasion by 48±28% (p<0.1) and KDM2A siRNA 2 by 64±16% (p<0.05) when compared to control siRNA. (D) Both KDM2A siRNA1 (100 pmol) and KDM2A siRNA2 (100 pmol) transfected MCF7 cells migrated into the wound in the presence of serum at 48 hr when compared to control siRNA transfected cells. Serum starved cells did not migrate into the wound with or without KDM2A suppression. (E) T47D cells additionally show significant migration into the wound after transfection with KDM2A siRNA1 and KDM2A siRNA 2 in the presence of serum at 24 hr when compared to control siRNA. Serum starved cells did not migrate into the wound with or without KDM2A suppression. Magnification-200X.</p

KDM2A co-localizes with E2F1 at 6 hr of serum stimulation.

<p>Quiescent MCF-7 cells were serum starved for 48 hr and serum stimulated for 6 hr and 18 hr. Cells were fixed, permeabilized for 5 min with 0.2% Triton X-100/PBS and immunostained for E2F1 (anti-mouse IgG, green) and KDM2A (anti-rabbit IgG, red). Cells were visualized by confocal microscopy. E2F1 was predominantly localized in the nucleus (upper panels), while KDM2A was more ubiquitously distributed in the cells (middle panels). Co-localization of E2F1 and KDM2A was observed in the nucleus at 6 hr of serum stimulation and disappeared totally by 18 hr (right panels). Images were captured at 630X oil using DM16000 inverted Leica TCS SP5 tandem scanning confocal microscope. Scale bar  = 200 µm. Pearson's correlation for co-localization at 6 hr was 1.0.</p

Silencing KDM2A enhances angiogenic tubulogenesis.

<p>(A) HUVEC cells were transfected with either control siRNA or KDM2A siRNA (75 pmol). 24 hr later, cells were plated on matrigel in complete media (asynchronous) or with or without 100 ng/ml VEGF. Images were captured 18 hr later using Leica inverted microscope and representative images are shown. (B) Tubule sprouting points were estimated using Image Pro software. Ablation of KDM2A showed significant increase (2-fold, p<0.01) in the number of sprouting points. (C) Real-Time showing increased mRNA expression of FLT-1 (2.2±0.11-fold, p<0.01) and KDR (1.7±0.9-fold, p<0.05) receptors upon silencing KDM2A expression. Simultaneous decrease in KDM2A mRNA levels (p<0.01) is seen with 75 pmol of KDM2A siRNA compared to control siRNA.</p