Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer death in the US. The protein kinase D (PKD) family has emerged as a promising target for cancer therapy with PKD1 being most intensively studied; however, its role in HNSCC has not been investigated. The expression of PKD was evaluated in human HNSCC by quantitative RT-PCR, Western blot and immunohistochemistry. Cell proliferation, wound healing, and matrigel invasion assays were performed upon siRNA-mediated knockdown of PKD1 in HNSCC cells, and subcutaneous xenograft mouse model was established by implantation of the stable doxycycline (Dox)-inducible PKD1 expression cell lines for analysis of tumorigenic activity in vivo. PKD1 was frequently downregulated in HNSCC cell lines at both transcript and protein levels. In human HNSCC tissues, PKD1 was significantly down-regulated in localized tumors and metastases, and in patient-paired tumor tissues as compared to their normal counterparts, which was in part due to epigenetic modification of the PRKD1 gene. The function of PKD1 in HNSCC was analyzed using stable doxycycline-inducible cell lines that express native or constitutive-active PKD1. Upon induction, the rate of proliferation, survival, migration and invasion of HNSCC cells did not differ significantly between the control and PKD1 overexpressing cells in the basal state, and depletion of endogenous PKD1 did not impact the proliferation of HNSCC cells. However, the median growth rate of the subcutaneous HNSCC tumor xenografts over time was elevated with PKD1 induction, and the final tumor weight was significantly increased in Dox-induced vs. the non-induced tumors. Moreover, induced expression of PKD1 promoted bombesin-induced cell proliferation of HNSCC and resulted in sustained ERK1/2 activation in response to gastrin-releasing peptide or bombesin stimulation, suggesting that PKD1 potentiates GRP/bombesin-induced mitogenic response through the activation of ERK1/2 in HSNCC cells. Our study has identified PKD1 as a frequently downregulated gene in HNSCC, and functionally, under certain cellular context, may play a role in GRP/bombesin-induced oncogenesis in HNSCC.
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.
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.
6033 Background: The epidermal growth factor receptor (EGFR) and Src family kinases are upregulated in HNSCC. EGFR interacts with cSrc to activate oncogenic STAT3 signaling; dual targeting is synergistic in HNSCC cell lines. In preclinical models, phosphorylated cSrc (pSrc) mediates resistance to erlotinib (E), a small molecule EGFR inhibitor. Baseline over-expression of pSrc was associated with E resistance in our prior window trial. Here, we conducted a four-arm phase 0 trial of E; dasatinib (D), an ATP-competitive inhibitor of cSrc; E+D; vs. placebo (P). Methods: Patients with operable Stage II-IVa HNSCC were randomized 1:1:1:1 to 7-21 days of neoadjuvant E 150 mg daily (n=11), D 100 mg daily (n=13), E+D (n=15), or P (for E; n=14). Paired tumor specimens were collected pre- and post-treatment. The primary endpoint, percent change in RECIST-measurable index lesions, was compared among groups by 2-way ANOVA. We analyzed the relationship between tumor percent change and pharmacodynamic expression of EGFR and cSrc pathway constituents. Pre-defined, hypothesis-driven analytes included pSTAT3, pSrc, pMet, and pMAPK. Results: From Apr 2009-Dec 2012, 58 patients were consented, 55 randomized, and 53 treated. Rash was observed on E arms; GI toxicities were observed in all active treatment groups. There was a significant decrease in tumor size in the E arms (p=0.0014), and no additive or independent effect from D (p=0.24). Among E-treated patients, high baseline pMAPK expression was associated with reduction in tumor size (p=0.03). An E-treated patient experiencing complete clinical response harbored a somatic MAPK1E322Kmutation. Among D-treated patients, high baseline pSTAT3 was associated with tumor progression (p=0.03). Conclusions: Brief neoadjuvant treatment with E significantly decreased tumor size in operable HNSCC, with no independent or added effect from D. Baseline pMAPK expression and genomic MAPK alterations warrant further study as response biomarkers for anti-EGFR therapy. High basal pSTAT3 in HNSCC patients may be independent of cSrc, explain therapeutic resistance, and preclude further development of D in biomarker-unselected cohorts. Clinical trial information: NCT00779389.
Abstract BACKGROUND Cetuximab combined with radiation therapy (RT) is an evidence‐based treatment for locally advanced head and neck squamous cell carcinoma (HNSCC); however, locoregional failure remains the primary cause of cancer‐related death in this disease. Intratumoral injection of epidermal growth factor receptor (EGFR)‐antisense plasmid DNA (EGFR‐AS) is safe and has been associated with promising lesional responses in patients who have recurrent/metastatic HNSCC. For the current study, the authors investigated the antitumor effects of cetuximab and EGFR‐AS in preclinical HNSCC models and reported their phase 1 experience adding intratumoral EGFR‐AS to cetuximab RT. METHODS Antitumor mechanisms were investigated in cell line and xenograft models. Phase 1 trial eligibility required stage IVA through IVC HNSCC and a measurable lesion accessible for repeat injections. Patients received standard cetuximab was for 9 weeks. EGFR‐AS was injected weekly until they achieved a lesional complete response. RT was delivered by conventional fractionation for 7 weeks, starting at week 3. Research biopsies were obtained at baseline and week 2. RESULTS When added to cetuximab, EGFR‐AS decreased cell viability and xenograft growth compared with EGFR‐sense control, partially mediated by reduced EGFR expression. Six patients were enrolled in the phase 1 cohort. No grade 2 or greater EGFR‐AS–related adverse events occurred. The best lesional response was a complete response (4 patients), and 1 patient each had a partial response and disease progression. EGFR expression decreased in 4 patients who had available paired specimens. CONCLUSIONS In preclinical models, dual EGFR inhibition with cetuximab and EGFR‐AS enhanced antitumor effects. In a phase 1 cohort, intratumoral EGFR‐AS injections, cetuximab, and RT were well tolerated. A phase 2 trial is needed to conduct an extended evaluation of safety and to establish efficacy.
Though there has been much advancement in treatment modalities, the prognosis for advanced pediatric solid malignancies continues to be poor. Leukopenia and immunosuppression are the commonest complications, which frequently lead to intermittent cessation of chemotherapy. This study was planned to evaluate the immunomodulatory efficacy and safety of Septilin syrup, as an adjunct, in pediatric solid malignancy patients undergoing chemotherapy. This study was a randomized, stratified, controlled phase III clinical trial and was approved by the Institutional Ethics Committee. Thirty-eight pediatric patients of advanced solid malignancy (Wilms’ tumor and neuroblastoma), in whom the diagnosis was confirmed by FNAC, and whose parents/guardians were willing to give informed written consent, were included in the study. Patients requiring immediate blood transfusion, and those who were unwilling to give informed written consent were excluded from the study. Informed written consent was obtained from parents/guardians of all the patients. All patients from the Septilin group received Septilin (5 ml, thrice daily), for 6 weeks, starting 48 hours prior to commencement of chemotherapy and the other group received only chemotherapy. At each follow-up visit, all patients were clinically evaluated for the response of chemotherapy, toxicity of chemotherapy, need for in-between postponement or stoppage of chemotherapy and requirement for blood transfusion during the course of chemotherapy. The immunological status was assessed by measuring the IgG, IgM and IgA levels, before initiation and on the ninth day of the chemotherapy. The predefined primary efficacy end points were improvement in immunoglobulin (IgG, IgM and IgA) levels and symptomatic improvement. The predefined secondary safety end points were reduced incidence of adverse reactions, reduced requirement of blood transfusion, reduction in chemotherapy, discontinuations and overall patient compliance to the drug treatment. Statistical analysis was done on an intention-to-treat basis.
