Beverly Wuertz

The Potential Role of Neutrophils in Promoting the Metastatic Phenotype of Tumors Releasing Interleukin-8

In the last decade, several groups have shown a direct correlation between the inappropriate or ectopic release of interleukin (IL)-8 by tumor cells in vitro and their growth and metastatic potential using in vivo models of tumor growth. IL-8 is a potent neutrophil chemoattractant. Neutrophils, as “early responders” to wounds and infections, release enzymes to remodel the extracellular matrix of the tissues through which they migrate to reach the site of the wound or infection. It is proposed that the host’s cellular response to IL-8 released by tumor cells enhances angiogenesis and contributes to tumor growth and progression. The activities released by the responding neutrophils could serve as enablers of tumor cell migration through the extracellular matrix, helping them enter the vasculature and journey to new, metastatic sites. The reactive oxygen species produced by neutrophilic oxidases to kill invading organisms have the potential to interact with tumor cells to attenuate their apoptotic cascade and increase their mutational rate. It is proposed that the increase in metastatic potential of tumors ectopically releasing IL-8 is, in part, attributable to their ability to attract neutrophils. Discussed here are possible mechanisms by which the neutrophils responding to ectopic IL-8 contribute to the in vivo growth, progression, and metastatic potential of tumor cells. Possible targets are also presented for the development of therapies to attenuate the effects of the ectopic IL-8 release by tumor cells.

A potential role for interleukin-8 in the metastatic phenotype of breast carcinoma cells.

This study shows a strong correlation between the metastatic potentials of breast carcinoma cell lines and their ectopic expression of interleukin-8 (IL-8). Correlations exist for both constitutive and induced levels of IL-8 released. A correlation was also observed between cell morphology, metastatic potential, and IL-8 profile. Metastatic lines are fusiform in appearance, whereas, nonmetastatic lines are epithelioid. The metastatic potential of two breast carcinoma lines was examined using an orthotopic model of spontaneous metastasis. Metastatic cells formed rapidly growing, poorly differentiated primary tumors that metastasized. Nonmetastatic cells formed rapidly growing differentiated primary tumors that did not produce detectable metastases. Comparison of IL-8 expression by the parental cells and cell cultures developed from primary and metastatic tumors, demonstrates that IL-8 released by cultured cells from the primary tumor is higher than that of the parental cells, and IL-8 released by cultured cells derived from the metastatic lung tumors is greater than that released by cultured cells derived from the primary tumor. These data demonstrate a strong correlation between the metastatic phenotype of a cell and its IL-8 expression, suggesting a role for IL-8 in promoting the metastatic potential of breast tumor cells.

Atypical methylation of the interleukin-8 gene correlates strongly with the metastatic potential of breast carcinoma cells

Previously, we have shown that a strong correlation exists between the metastatic potential of breast carcinoma cell lines and their ectopic expression of IL-8. The undifferentiated, highly metastatic cell lines with high metastatic potential produce much more IL-8 than their differentiated lower metastatic counterparts. After eliminating the possibility that transcription factor activity was responsible for differences in IL-8 release, we examined the IL-8 gene for possible epigenetic modifications. Here, we report an aberrant methylation pattern that may be responsible for the differences in IL-8 release between the high and low metastatic cell lines. We determined that none of the deoxycytidylate-phosphate-deoxyguanylate (CpG) sites in the reported IL-8 promoter were methylated in either cell type. Much further upstream in the IL-8 gene, two CpG sites were identified that are differentially methylated. These two sites were fully methylated in the high metastatic cell lines, which produce large quantities of IL-8 and remain unmethylated in the low metastatic cell lines where the IL-8 gene is relatively silent. The DNA methylation results presented here differ from the common epigenetic paradigm in which methylation of promoter CpG islands silences gene expression, suggesting that there are additional epigenetic control mechanisms that as yet have not been fully appreciated or explored.

Cigarette Smoke Activates NFκB and Induces Muc5b Expression in Mouse Middle Ear Cells

Objectives: Cigarette smoke exposure is a significant risk factor in the development of otitis media (OM). Nuclear factor kappa B (NF‐κB) is a ubiquitous transcription factor known to mediate cigarette smoke effects on gene regulation in multiple cell types. The MUC5B mucin gene contains several putative NF‐κB sites in its promoter and is the predominant mucin expressed in human OM. We hypothesized that in vitro stimulation of a recently developed model system, murine middle ear epithelial cells (MEEC), with cigarette smoke condensate (CSC) activates NF‐κB and subsequently induces Muc5b gene expression.

Tobacco carcinogen mediated up-regulation of AP-1 dependent pro-angiogenic cytokines in head and neck carcinogenesis

Tobacco is notably genotoxic and associated with head and neck carcinogenesis. Cigarette carcinogens have the capacity to alter early response gene expression in tobacco‐related malignancies via genes such as nuclear factor kappa B (NFκB). A number of early response gene activation events are also facilitated by fos/jun activator protein 1 (AP‐1) associated pathways. In the present study, we hypothesize that tobacco products may induce microenvironment alterations, promoting angiogenesis and providing a permissive environment for head and neck cancer progression. In an in vitro analysis, we employed immortalized oral keratinocyte (HOK‐16B) and laryngeal squamous carcinoma (UM‐SCC‐11A) cells to investigate interleukin (IL)‐8 and vascular endothelial growth factor (VEGF) induction by cigarette smoke condensate (CSC). IL‐8 and VEGF expression is based on interactions between NFκB, AP‐1, and NF‐IL6. We identified at least 1.5‐fold dose‐dependent induction of AP‐1, VEGF, and IL‐8 promoter/reporter gene activity after 24 h exposure to CSC. Next, we stably transfected UM‐SCC‐11A cells with A‐Fos, a dominant negative AP‐1 protein. Treatment with CSC of the A‐Fos cell lines compared to empty vector controls significantly down‐regulated AP‐1, VEGF, and IL‐8 promoter/reporter gene expression. We also performed ELISAs and discovered significant up‐regulation of IL‐8 and VEGF secretion by UMSCC 11A after treatment with phorbol 12‐myristate 13‐acetate, tumor necrosis factor alpha, and CSC, which was down‐regulated by the A‐Fos dominant negative protein. We conclude tobacco carcinogens up‐regulate AP‐1 activity and AP‐1 dependent IL‐8 and VEGF gene expression in head and neck cancer. This up‐regulation may promote an angiogenic phenotype favoring invasion in both premalignant and squamous cancer cells of the head and neck.

TNF‐α Drives Matrix Metalloproteinase‐9 in Squamous Oral Carcinogenesis

Objectives/Hypothesis: It is well known that invasion is a seminal event in the progression of oral and other head and neck carcinoma sites. We have previously demonstrated tumor necrosis factor (TNF)‐α and its dependent cytokines are upregulated in saliva during oral carcinogenesis. TNF‐dependent events stimulate nuclear factor (NF)‐κB and many NF‐κB‐dependent genes are associated with cancer progression.

Cigarette smoke condensate induces nuclear factor kappa-b activity and proangiogenic growth factors in aerodigestive cells.

Aerodigestive cancer risk of both lung and head and neck cancers has been linked to the genotoxic effects of tobacco use. These effects include upregulation of nuclear factor kappa‐B (NFκB) and its downstream products associated with both lung and head and neck cancer malignant progression.

PPAR activation and decreased proliferation in oral carcinoma cells with 4-HPR.

OBJECTIVE: To explore whether the mechanism of action of 4-hydroxyphenylretinamide (4-HPR, fenretidine), a synthetic retinoid, involves the functional activation of the nuclear hormone receptor class known as PPARs (peroxisome proliferator-activated receptors). Also, to examine whether anti-proliferative effects of this agent in head and neck cancer cells occur at biologically relevant concentrations. STUDY DESIGN/METHODS: CA 9–22, NA, and UM SCC 11B cells were treated with 4-HPR during their log phase growth and functional activation of PPAR γ was evaluated by plate luminometry. Cellular proliferation was analyzed by standard MTT cell proliferation assays and cell counting. Students t tests were performed for all experiments. RESULTS: Significant dose-dependent increases in PPAR γ activation occurred in response to 4-HPR treatment. Proliferation was significantly inhibited by 4-HPR in a dose-dependent manner as judged by MTT and cell counting assays. These effects occurred at equimolar concentrations in both types of experiments within a range of clinically achievable doses (1–4 μM) of 4-HPR. CONCLUSIONS: 4-HPR can functionally activate PPAR γ at clinically achievable doses. Decreased cancer cell proliferation secondary to PPAR γ activation has been observed in other malignancies as well as upper aerodigestive cancer. PPAR γ activation by 4-HPR represents another potential anti-cancer mechanism of action for this drug. CLINICAL SIGNIFICANCE: PPAR γ activation represents a novel target for anti-cancer therapy for head and neck cancer and the current level of clinical toxicity of 4-HPR would be judged acceptable to utilize this agent alone or in combination chemotherapy.

Quantitative Proteomic Analysis of Oral Brush Biopsies Identifies Secretory Leukocyte Protease Inhibitor as a Promising, Mechanism-Based Oral Cancer Biomarker

A decrease in the almost fifty percent mortality rate from oral cancer is needed urgently. Improvements in early diagnosis and more effective preventive treatments could affect such a decrease. Towards this end, we undertook for the first time an in-depth mass spectrometry-based quantitative shotgun proteomics study of non-invasively collected oral brush biopsies. Proteins isolated from brush biopsies from healthy normal tissue, oral premalignant lesion tissue (OPMLs), oral squamous cell carcinoma (OSCC) and matched control tissue were compared. In replicated proteomic datasets, the secretory leukocyte protease inhibitor (SLPI) protein stood out based on its decrease in abundance in both OPML and OSCC lesion tissues compared to healthy normal tissue. Western blotting in additional brushed biopsy samples confirmed a trend of gradual decreasing SLPI abundance between healthy normal and OPML tissue, with a larger decrease in OSCC lesion tissue. A similar SLPI decrease was observed in-vitro comparing model OPML and OSCC cell lines. In addition, exfoliated oral cells in patients’ whole saliva showed a loss of SLPI correlated with oral cancer progression. These results, combined with proteomics data indicating a decrease in SLPI in matched healthy control tissue from OSCC patients compared to tissue from healthy normal tissue, suggested a systemic decrease of SLPI in oral cells correlated with oral cancer development. Finally, in-vitro experiments showed that treatment with SLPI significantly decreased NF-kB activity in an OPML cell line. The findings indicate anti-inflammatory activity in OPML, supporting a mechanistic role of SLPI in OSCC progression and suggesting its potential for preventative treatment of at-risk oral lesions. Collectively, our results show for the first time the potential for SLPI as a mechanism-based, non-invasive biomarker of oral cancer progression with potential in preventive treatment.

The Effect of Indomethacin on Paclitaxel Sensitivity and Apoptosis in Oral Squamous Carcinoma Cells: The Role of Nuclear Factor–κB Inhibition

OBJECTIVE To investigate new strategies to intensify chemosensitivity in head and neck squamous cell carcinoma. DESIGN Oral squamous carcinoma cells were examined for nuclear factor-κB (NF-κB) activation and binding activity by paclitaxel, an agent currently used in head and neck cancer chemotherapy. Electromobility shift assays were used to assess the effect of indomethacin on NF-κB binding activity. Cell proliferation assays were used to study cell sensitivity to paclitaxel. To examine whether cytotoxicity could be increased by specifically inhibiting NF-κB, a dominant negative cell line, inhibitor kappa B-alpha (IκBα), was stably expressed in CA-9-22 cells. RESULTS Paclitaxel possessed the capacity to functionally activate NF-κB, as demonstrated by luciferase reporter gene assays and electromobility shift assay. Indomethacin was able to inhibit paclitaxel-mediated NF-κB activation and promote apoptosis of paclitaxel-treated cells at 24 hours. Indomethacin augmented the paclitaxel cell-killing effect. The dominant negative IκBα cell line exhibited increased chemosensitization to paclitaxel by 2- to 10-fold. CONCLUSIONS Paclitaxel has the capacity to activate NF-κB in oral squamous carcinoma cells. Indomethacin can reverse this activation to decrease cell proliferation and increase apoptosis. Treatment strategies that combine paclitaxel with indomethacin may have therapeutic benefits attributable to paclitaxel chemosensitization through NF-κB inhibition.