Emily E. Helman

Anti-EMMPRIN Monoclonal Antibody as a Novel Agent for Therapy of Head and Neck Cancer

Purpose: Extracellular matrix metalloprotease inducer (EMMPRIN) is a tumor surface protein that promotes growth and is overexpressed in head and neck cancer. These features make it a potential therapeutic target for monoclonal antibody (mAb)–based therapy. Because molecular therapy is considered more effective when delivered with conventional cytotoxic agents, anti-EMMPRIN therapy was assessed alone and in combination with external beam radiation. Experimental Design: Using a murine flank model, loss of EMMPRIN function was achieved by transfection with a small interfering RNA against EMMPRIN or treatment with a chimeric anti-EMMPRIN blocking mAb. Cytokine expression was assessed for xenografts, tumor cells, fibroblasts, and endothelial cells. Results: Animals treated with anti-EMMPRIN mAb had delayed tumor growth compared with untreated controls, whereas treatment with combination radiation and anti-EMMPRIN mAb showed the greatest reduction in tumor growth (P = 0.001). Radiation-treated EMMPRIN knockdown xenografts showed a reduction in tumor growth compared with untreated knockdown controls (P = 0.01), whereas radiation-treated EMMPRIN–expressing xenografts did not show a delay in tumor growth. Immunohistochemical evaluation for Ki67 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) resulted in a reduction in proliferation (P = 0.007) and increased apoptosis in anti-EMMPRIN mAb–treated xenografts compared with untreated controls (P = 0.087). In addition, we provide evidence that EMMPRIN suppression results in decreased interleukin 1β (IL-1β), IL-6, and IL-8 cytokine production, in vitro and in vivo. Conclusions: These data suggest that anti-EMMPRIN antibody inhibits tumor cell proliferation in vivo and may represent a novel targeted treatment option in head and neck squamous cell carcinoma.

Disruption of the AKT pathway inhibits metastasis in an orthotopic model of head and neck squamous cell carcinoma

MK‐2206 is an orally active, allosteric inhibitor of AKT, a component of the phosphatidylinositol‐3 kinase (PI3K) pathway. The PI3K‐AKT pathway is a downstream signaling pathway that has recently been found to play an important role in head and neck squamous cell carcinoma (HNSCC). The objective of this study is to examine the role AKT inhibition may play in treatment of HNSCC.

Optical imaging predicts tumor response to anti-EGFR therapy.

To evaluate cetuximab treatment in head and neck squamous cell carcinoma xenografts and cell lines, we investigated a preclinical model of head and neck squamous cell carcinoma. Head and neck squamous cell carcinoma cell lines SCC-1, FaDu, CAL27, UM-SCC-5 and UM-SCC-22A were used to generate subcutaneous flank xenografts in SCID mice. Mice were divided into control and cetuximab treatment groups, mice in the latter group received 250 μg cetuximab once weekly for four weeks. After completion of therapy, SCC-1 (P < 0.001), UM-SCC-5 (P < 0.001), UM-SCC-22A (P = 0.016) and FaDu (P = 0.007) tumors were significantly smaller than control, while CAL27 tumors were not different from controls (P = 0.90). Mice were systemically injected with 50 μg of the Cy5.5-cetuximab bioconjugate and imaged by stereomicroscopy to determine if tumor fluorescence predicted tumor response. Intact tumor fluorescence did not predict response. Tissue was harvested from untreated xenografts to evaluate ex vivo imaging. Cell lines were then evaluated in vitro for fluorescence imaging after Cy5.5-cetuximab bioconjugate labeling. The location of fluorescence observed in labeled cells was significantly different for cell lines that responded to treatment, relative to unresponsive cells. Tumors from cell lines that showed low internalized signal in vitro responded best to treatment with cetuximab. This preclinical model may aid in determining which cancer patients are best suited for cetuximab therapy.

EGFR expression in advanced head and neck cutaneous squamous cell carcinoma

The significance of epidermal growth factor receptor (EGFR) expression in advanced cutaneous squamous cell carcinoma (SCC) of the head and neck remains poorly understood.

Anti-EMMPRIN antibody treatment of head and neck squamous cell carcinoma in an ex-vivo model

Targeting the molecular pathways associated with carcinogenesis remains the greatest opportunity to reduce treatment-related morbidity and mortality. Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as CD147, is a cell surface molecule known to promote tumor growth and angiogenesis in preclinical studies of head and neck carcinoma making it an excellent therapeutic target. To evaluate the feasibility of anti-EMMPRIN therapy, an ex-vivo human head and neck cancer model was established using specimens obtained at the time of surgery (n=22). Tumor slices were exposed to varying concentrations of anti-EMMPRIN monoclonal antibody and cetuximab for comparison purposes. Cetuximab is the only monoclonal antibody currently approved for the treatment of head and neck carcinoma. After treatment, tumor slices were assessed by immunohistochemistry and western blot analysis for apoptosis (TUNEL) and EMMPRIN expression. Of the tumor specimens 33% showed a significant reduction in mean ATP levels after treatment with cetuximab compared with untreated controls, whereas 58% of the patients responded to anti-EMMPRIN therapy (P<0.05). Samples, which showed reactivity to anti-EMMPRIN, also had greater EMMPRIN expression based on immunohistochemistry staining (49%) when compared with nonresponders (25%, P=0.06). In addition, TUNEL analysis showed a larger number of cells undergoing apoptosis in antibody-treated tumor slices (77%) compared with controls (30%, P<0.001) with activation of apoptotic proteins, caspase 3 and caspase 8. This study shows the potential of anti-EMMPRIN to inhibit proliferation and promote apoptosis and suggests its future role in the targeted treatment of head and neck carcinoma.

Extracelluar matrix metalloproteinase as a novel target for pancreatic cancer therapy.

The objective of this study was to evaluate extracellular matrix metalloproteinase (EMMPRIN) as a novel target in orthotopic pancreatic cancer murine models. MIA PaCa-2 human pancreatic tumor cells were implanted in groups 1 and 3–7, whereas MIA PaCa-2 EMMPRIN knockdown cells were implanted in group 2. Dosing with anti-EMMPRIN antibody started immediately after implantation for groups 1–3 (residual tumor model) and at 21 days after cell implantation for groups 4–7 (established tumor model). Groups 3, 5, and 7 were treated with anti-EMMRPIN antibody (0.2–1.0 mg) twice weekly for 2–3 weeks, whereas the other groups served as the control. In the residual tumor model, tumor growth of anti-EMMPRIN-treated group was successfully arrested for 21 days (15±4 mm3), which was significantly lower than that of the EMMPRIN knockdown group (80±15 mm3; P=0.001) or the control group (240±41 mm3; P<0.001). In the established tumor model, anti-EMMPRIN therapy lowered tumor volume increase by approximately 40% compared with the control, regardless of the dose amount. Ki67-expressed cell density of group 5 was 939±150 mm−2, which was significantly lower than that of group 4 (1709±145 mm−2; P=0.006). Microvessel density of group 5 (30±6 mm−2) was also significantly lower than that of group 4 (53±5 mm−2; P=0.014), whereas the microvessel size of group 5 (191±22 &mgr;m2) was significantly larger than that of group 4 (113±26 &mgr;m2; P=0.049). These data show the high potential of anti-EMMPRIN therapy for pancreatic cancer and support its clinical translation.

EMMPRIN expression is required for response to bevacizumab therapy in HNSCC xenografts

The HNSCC cell line, FaDu was stably transfected with control vector (FaDu) or with plasmid expressing small interfering RNA against EMMPRIN (FaDu/siE). Tumor cells were treated with bevacizumab (0, 25, 50, and 75 ng/ml) in vitro, and then cell counts were performed at 72 h. For in vivo analysis, tumor cells were xenografted onto the flank of SCID mice, and were treated with 100 microg bevacizumab twice weekly for three weeks. Xenograft samples from the control and treatment groups were analyzed for microvessel density. Escalating doses of bevacizumab had no effect on the growth of tumor cells in vitro (P.or=0.086). However, tumor xenografts expressing EMMPRIN responded to bevacizumab treatment (P=0.0013), whereas the EMMPRIN knockdown cell line did not (P=0.7942). Immunohistochemical analysis demonstrated that microvascular density was reduced in the treated FaDu tumors (P=0.005), but not in the FaDu/siE tumors (P=0.48). Currently there is limited information on biomarkers to predict response to bevacizumab. By demonstrating effectiveness of bevacizumab therapy in tumors that express EMMPRIN, but not in tumors with silenced EMMPRIN expression, this study suggests that EMMPRIN may serve as a biomarker for response to bevacizumab treatment.

Anti-EMMPRIN treatment of HNSCC in an ex vivo model.

Objective: Targeting the molecular pathways associated with carcinogenesis remains the greatest opportunity to reduce treatment related morbidity and mortality. Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as CD147, is a cell surface molecule known to promote tumor growth and angiogenesis in preclinical studies of head and neck carcinoma. To evaluate the feasibility of anti-CD147 therapy, an ex vivo human head and neck cancer model was established.

Abstract 4575: Novel monoclonal antibody targeting EMMPRIN for pancreatic cancer therapy

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose: To evaluate a novel monoclonal antibody targeting extracelluar matrix metalloproteinase (EMMPRIN) in orthotopic pancreatic-cancer murine models. Methods: In vitro cytotoxic assay of anti-EMMPRIN mAb was conducted with MIA PaCA-2 and PANC-1 human pancreatic-cancer cell lines. Binding assay of Tc-99m labeled anti-EMMPRIN mAb was repeated three times independently for MIA PaCa-2 cells or MIA PaCa-2 cells silenced for EMMPRIN gene expression by siRNA. For the in vivo animal study, MIA PaCa-2 cells were implanted in groups 1-6, while EMMPRIN silenced cells were implanted in group 7. For groups 1-4 (solid tumor model, n=5-7/group), dosing started at 21 days after cell implantation, while dosing started immediately after cell implantation for groups 5-7 (residual tumor model, n=10-12/group); groups 1 and 2 were injected with PBS and anti-EMMPRIN mAB (0.2mg) twice weekly for 3 weeks respectively; ultrasound imaging (USI) was applied at days 21, 28, 35, and 42 to determine tumor volume. Groups 3 and 4 were injected with PBS and anti-EMMPRIN mAb (1mg) twice weekly for 2 weeks, respectively; USI and diffusion-weighted MRI were applied at days 21, 28, and 35. Groups 5 and 6 were injected with PBS and anti-EMMPRIN mAB (0.2mg) twice weekly for 3 weeks respectively; group 7 was injected with PBS during the same time. For groups 5-7, USI was applied at 15 and 21 days post cell injection. Ki-67 and TUNEL staining were conducted on tumor sections from groups 1 and 2 at the study end. Results: In vitro treatment with antibody alone did not demonstrate significant cytotoxic effect in either cell line. The Kd in MIA PaCa-2 cells was 4.31±0.59 (mean ± SE) nM, which was not statistically different from that in EMMPRIN silenced cells. The average number of EMMPRIN per MIA PaCa-2 cell was 582,000±56,000, significantly higher than that per EMMPRIN silenced cells at 220,000±89,000 (p=0.03). Tumor-volume increases of group 1 relative to day 21 were 282±33, 495±107, and 725±166% at days 28, 35, and 42, while those of group 2 were 191±7, 263±27, and 429±39%; groups 1 and 2 were statistically different at day 28 (p=0.03), not at the other days. Group 3 showed an increase in tumor-volume of 317±55 and 505±96% at days 28 and 35 respectively, while those in group 4 were 189±27 and 244±41%; groups 3 and 4 were statistically different at day 35 (p=0.04), but not at day 28. Mean intra-tumoral ADC change of group 4 was not statistically significant from that of group 3. Tumor volumes of groups 5-7 were 66±17, 21±5, and 4±2mm3 at day 15, while those at day 21 were 240±41, 80±15, and 15±4mm3, which represent statistical differences among groups at each day (p<0.05). Proliferating cell density of group 2 was significantly lower than that of group 1 (p=0.01), while apoptotic cell densities were not. Conclusion: Tumor growth was successfully arrested following treatment with anti-EMMPRIN antibody for both solid- and residual-tumor models, consistent with reduced proliferating cell density. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4575. doi:10.1158/1538-7445.AM2011-4575

R422 – EMMPRIN as a Novel Target in Head and Neck Cancer

Problem To determine if blocking EMMPRIN function in vivo inhibits tumor growth alone or when combined with radiation therapy in a preclinical model of head and neck squamous cell carcinoma (HNSCC). Methods Immunodeficient mice bearing SCC-1 subcutaneous xenografts were sorted into four treatment groups: anti-EMMPRIN antibody alone, anti-EMMPRIN antibody in combination with radiotherapy, radiotherapy alone, and untreated control groups (n = 7 per group). Each animal in the treatment groups received a total dose of 1.2 mg of antibody and/or 12 Gy of 60Co radiation, biweekly over three weeks. To investigate possible mechanisms of action for anti-EMMPRIN therapy, xenograft samples from the control group and antibody alone group were analyzed for proliferation (Ki67) and apoptosis (TUNEL). Furthermore, the expressions of EMMPRIN induced cytokines were assessed by ELISA, including IL-1?, IL-6, IL-8, and VEGF. Results Two weeks after completion of treatment, mean tumor surface area for the antibody alone group was smaller than the untreated group (P = 0.0006), as was the radiation alone group (P = 0.0029), while combination therapy yielded optimal results (P = 0.0003). Anti-EMMPRIN antibody treated tumors had a significant reduction in proliferation (P = 0.007), and analysis of in vivo apoptosis by TUNEL assay showed a higher number of cells undergoing apoptosis in the treatment group (18%) vs. untreated (5%; P = 0.087), although this failed to reach statistical significance. Treatment of xenografted tumors resulted in decreased expression of IL-1? (P = 0.0079), IL-6 (P = 0.077), IL-8 (P= 0.1481), and VEGF (P = 0.0538) when compared to animals receiving no treatment. Conclusion This data suggests that blocking EMMPRIN function in vivo inhibits tumor cell proliferation and sensitizes tumor xenografts to radiation in vivo. Significance EMMPRIN may represent a novel target for treatment in head and neck cancer. Support This work was supported by grants from the National Cancer Institute (NCI K08CA102154) and the National Institute of Health (2T32 CA091078-06).