Malabika Sen

Targeting Stat3 abrogates EGFR inhibitor resistance in cancer.

Purpose: EGF receptor (EGFR) is upregulated in most epithelial cancers where signaling through EGFR contributes to cancer cell proliferation and survival. The limited clinical efficacy of EGFR inhibitors suggests that identification of resistance mechanisms may identify new pathways for therapeutic targeting. STAT3 is upregulated in many cancers and activated via both EGFR-dependent and -independent pathways. In the present study, we tested the consequences of STAT3 inhibition in EGFR inhibitor–resistant head and neck squamous cell carcinoma (HNSCC) and bladder cancer models to determine whether STAT3 blockade can enhance responses to EGFR targeting. Experimental Design: pSTAT3 expression was assessed in human HNSCC tumors that recurred following cetuximab treatment. Cetuximab-sensitive and -resistant cell lines were treated with a STAT3 decoy to determine EC50 concentrations and the effects on STAT3 target gene expression by Western blotting. In vivo assays included evaluation of antitumor efficacy of STAT3 decoy in cetuximab-sensitive and -resistant models followed by immunoblotting for STAT3 target protein expression. Results: Targeting STAT3 with a STAT3 decoy reduced cellular viability and the expression of STAT3 target genes in EGFR inhibitor resistance models. The addition of a STAT3 inhibitor to EGFR blocking strategies significantly enhanced antitumor effects in vivo. Biopsies from HNSCC tumors that recurred following cetuximab treatment showed increased STAT3 activation compared with pretreatment biopsies. Conclusions: These results suggest that STAT3 activation contributes to EGFR inhibitor resistance both in HNSCC and bladder cancer where concomitant targeting of STAT3 may represent an effective treatment strategy. Clin Cancer Res; 18(18); 4986–96. ©2012 AACR.

Guggulsterone enhances head and neck cancer therapies via inhibition of signal transducer and activator of transcription-3

Treatment of human head and neck squamous cell carcinoma (HNSCC) cell lines with guggulsterone, a widely available, well-tolerated nutraceutical, demonstrated dose-dependent decreases in cell viability with EC(50)s ranging from 5 to 8 microM. Guggulsterone induced apoptosis and cell cycle arrest, inhibited invasion and enhanced the efficacy of erlotinib, cetuximab and cisplatin in HNSCC cell lines. Guggulsterone induced decreased expression of both phosphotyrosine and total signal transducer and activator of transcription (STAT)-3, which contributed to guggulsterones growth inhibitory effect. Hypoxia-inducible factor (HIF)-1alpha was also decreased in response to guggulsterone treatment. In a xenograft model of HNSCC, guggulsterone treatment resulted in increased apoptosis and decreased expression of STAT3. In vivo treatment with a guggulsterone-containing natural product, Guggulipid, resulted in decreased rates of tumor growth and enhancement of cetuximabs activity. Our results suggest that guggulsterone-mediated inhibition of STAT3 and HIF-1alpha provide a biologic rationale for further clinical investigation of this compound in the treatment of HNSCC.

Combined Targeting of Epidermal Growth Factor Receptor, Signal Transducer and Activator of Transcription-3, and Bcl-XL Enhances Antitumor Effects in Squamous Cell Carcinoma of the Head and Neck

Squamous cell carcinoma of the head and neck (SCCHN) is a leading cause of cancer deaths worldwide. Epidermal growth factor receptor (EGFR), an upstream mediator of signal transducer and activator of transcription (STAT)-3 is overexpressed in a variety of cancers, including SCCHN. Therapies such as monoclonal antibodies and tyrosine kinase inhibitors targeting EGFR have demonstrated limited antitumor efficacy, which may be explained, in part, by persistent STAT3 activation despite EGFR inhibition. STAT3 activation induces expression of target genes in SCCHN, including Bcl-XL, a mediator of antiapoptotic activity. Bcl-XL is commonly overexpressed in SCCHN where it correlates with chemoresistance, making it a potential therapeutic target. Targeting the EGFR-STAT3-Bcl-XL pathway at several levels, including the upstream receptor, the intracellular transcription factor, and the downstream target gene, has not been investigated previously. Using erlotinib, an EGFR-specific reversible tyrosine kinase inhibitor in combination with a STAT3 transcription factor decoy, we found enhanced antitumor effects in vitro and in vivo. The combination of the STAT3 decoy and gossypol, a small molecule targeting Bcl-XL, also yielded enhanced inhibition of cell proliferation. The triple combination of erlotinib, STAT3 decoy, and gossypol further enhanced cell growth inhibition and apoptosis in vitro, and it down-regulated signaling molecules further downstream of the EGFR-STAT3 signaling pathway, such as cyclin D1. These results suggest that combined targeting of several components of an oncogenic signaling pathway may be an effective therapeutic strategy for SCCHN.

Epidermal growth factor receptor variant III mediates head and neck cancer cell invasion via STAT3 activation

Epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) where aberrant signaling downstream of this receptor contributes to tumor growth. EGFR variant III (EGFRvIII) is the most commonly altered form of EGFR and contains a truncated ligand-binding domain. We previously reported that EGFRvIII is expressed in up to 40% of HNSCC tumors where it is associated with increased proliferation, tumor growth and chemoresistance to antitumor drugs including the EGFR-targeting monoclonal antibody cetuximab. Cetuximab was FDA-approved in 2006 for HNSCC but has not been shown to prevent invasion or metastasis. This study was undertaken to evaluate the mechanisms of EGFRvIII-mediated cell motility and invasion in HNSCC. We found that EGFRvIII induced HNSCC cell migration and invasion in conjunction with increased signal transducer and activator of transcription 3 (STAT3) activation, which was not abrogated by cetuximab treatment. Further investigation showed that EGF-induced expression of the STAT3 target gene HIF1-α, was abolished by cetuximab in HNSCC cells expressing wild-type EGFR under hypoxic conditions, but not in EGFRvIII-expressing HNSCC cells. These results suggest that EGFRvIII mediates HNSCC cell migration and invasion by increased STAT3 activation and induction of HIF1-α, which contribute to cetuximab resistance in EGFRvIII-expressing HNSCC tumors.

Bortezomib up-regulates activated signal transducer and activator of transcription-3 and synergizes with inhibitors of signal transducer and activator of transcription-3 to promote head and neck squamous cell carcinoma cell death

Head and neck squamous cell carcinomas (HNSCC) are commonly resistant to conventional chemotherapy drugs and exhibit overexpression of signal transducer and activator of transcription 3 (STAT3). STAT3 promotes both the proliferation and survival of HNSCC cells. Recent studies have shown that the proteasome inhibitor bortezomib shows cytotoxic activity against HNSCC in vitro and in vivo. We report that treatment of HNSCC cells with bortezomib led to up-regulation of total STAT3 protein and the phosphorylated/activated form of STAT3, as well as an increase in cellular STAT3 activity. This suggested that the ability of bortezomib to kill HNSCC cells may be blunted due to induction of STAT3, and inhibition of STAT3 may be a useful means for improving bortezomib efficacy. Indeed, forced expression of dominant-active STAT3 inhibited bortezomib-induced cell death, whereas expression of dominant-negative STAT3 served to enhance killing by this compound. In addition, specific inhibition of STAT3 with the use of a STAT3 decoy oligonucleotide resulted in enhancement of bortezomib-induced apoptosis signaling and loss of clonogenic survival. Cotreatment of HNSCC cells with bortezomib and guggulsterone, a naturally occurring compound known to inhibit STAT3 activation, led to synergistic activation of cell death and loss of clonogenic survival. In summary, these studies show that bortezomib induces the expression of active STAT3, a key growth- promoting protein in HNSCC cells. Furthermore, our findings suggest that the therapeutic activity of bortezomib against HNSCC may be markedly improved by cotreatment with molecular targeting agents against STAT3. [Mol Cancer Ther 2009;8(8):2211–20]

PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response

Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment.

Nucleic acid-based approaches to STAT inhibition

Silencing of abnormally activated genes can be accomplished in a highly specific manner using nucleic acid based approaches. The focus of this review includes the different nucleic acid based inhibition strategies such as antisense oligodeoxynucleotides, small interfering RNA (siRNA), dominant-negative constructs, G-quartet oligonucleotides and decoy oligonucleotides, their mechanism of action and the effectiveness of these approaches to targeting the STAT (signal transducer and activator of transcription) proteins in cancer. Among the STAT proteins, especially STAT3, followed by STAT5, are the most frequently activated oncogenic STATs, which have emerged as plausible therapeutic cancer targets. Both STAT3 and STAT5 have been shown to regulate numerous oncogenic signaling pathways including proliferation, survival, angiogenesis and migration/invasion.

JAK Kinase Inhibition Abrogates STAT3 Activation and Head and Neck Squamous Cell Carcinoma Tumor Growth

Aberrant activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) 3 has been implicated in cell proliferation and survival of many cancers including head and neck squamous cell carcinoma (HNSCC). AZD1480, an orally active pharmacologic inhibitor of JAK1/JAK2, has been tested in several cancer models. In the present study, the in vitro and in vivo effects of AZD1480 were evaluated in HNSCC preclinical models to test the potential use of JAK kinase inhibition for HNSCC therapy. AZD1480 treatment decreased HNSCC proliferation in HNSCC cell lines with half maximal effective concentration (EC50) values ranging from 0.9 to 4 μM in conjunction with reduction of pSTAT3Tyr705 expression. In vivo antitumor efficacy of AZD1480 was demonstrated in patient-derived xenograft (PDX) models derived from two independent HNSCC tumors. Oral administration of AZD1480 reduced tumor growth in conjunction with decreased pSTAT3Tyr705 expression that was observed in both PDX models. These findings suggest that the JAK1/2 inhibitors abrogate STAT3 signaling and may be effective in HNSCC treatment approaches.

Caspase-8 mutations in head and neck cancer confer resistance to death receptor-mediated apoptosis and enhance migration, invasion, and tumor growth.

Little is known regarding molecular markers in head and neck squamous cell carcinoma (HNSCC) that predict responsiveness to different therapeutic regimens or predict HNSCC progression. Mutations in procaspase‐8 occur in 9% of HNSCC primary tumors, but the functional consequences of these mutations are poorly understood. In this study, we examined the impact of four, representative, HNSCC‐associated procaspase‐8 mutations on activation of the extrinsic apoptosis pathway, as well as cellular migration and invasion, and in vivo tumor growth. All four mutant proteins acted to potently inhibit activation of apoptosis following treatment with TRAIL or agonistic anti‐Fas. In contrast to wild‐type procaspase‐8, the mutant proteins were not recruited to FADD following treatment with TRAIL or anti‐Fas, but may be constitutively bound by FADD. Three of the four procaspase‐8 mutants promoted enhanced cellular migration and invasion through matrigel, relative to that seen with the wild‐type procaspase‐8 protein. Procaspase‐8 mutation also stimulated the growth of HNSCC xenograft tumors. These findings indicate that HNSCC‐associated procaspase‐8 mutations inhibit activation of the extrinsic apoptosis pathway and are likely to represent markers for resistance to therapeutic regimens incorporating death receptor activators. Moreover, procaspase‐8 mutations may serve as markers of HNSCC tumor progression, as exemplified by enhanced migration, invasion, and tumor growth.

Prevention of Carcinogen-Induced Oral Cancer by Sulforaphane

Chronic exposure to carcinogens represents the major risk factor for head and neck squamous cell carcinoma (HNSCC). Beverages derived from broccoli sprout extracts (BSE) that are rich in glucoraphanin and its bioactive metabolite sulforaphane promote detoxication of airborne pollutants in humans. Herein, we investigated the potential chemopreventive activity of sulforaphane using in vitro models of normal and malignant mucosal epithelial cells and an in vivo model of murine oral cancer resulting from the carcinogen 4-nitroquinoline-1-oxide (4NQO). Sulforaphane treatment of Het-1A, a normal mucosal epithelial cell line, and 4 HNSCC cell lines led to dose- and time-dependent induction of NRF2 and the NRF2 target genes NQO1 and GCLC, known mediators of carcinogen detoxication. Sulforaphane also promoted NRF2-independent dephosphorylation/inactivation of pSTAT3, a key oncogenic factor in HNSCC. Compared with vehicle, sulforaphane significantly reduced the incidence and size of 4NQO-induced tongue tumors in mice. A pilot clinical trial in 10 healthy volunteers evaluated the bioavailability and pharmacodynamic activity of three different BSE regimens, based upon urinary sulforaphane metabolites and NQO1 transcripts in buccal scrapings, respectively. Ingestion of sulforaphane-rich BSE demonstrated the greatest, most consistent bioavailability. Mucosal bioactivity, defined as 2-fold or greater upregulation of NQO1 mRNA, was observed in 6 of 9 evaluable participants ingesting glucoraphanin-rich BSE; 3 of 6 ingesting sulforaphane-rich BSE; and 3 of 9 after topical-only exposure to sulforaphane-rich BSE. Together, our findings demonstrate preclinical chemopreventive activity of sulforaphane against carcinogen-induced oral cancer, and support further mechanistic and clinical investigation of sulforaphane as a chemopreventive agent against tobacco-related HNSCC. Cancer Prev Res; 9(7); 547–57. ©2016 AACR.