Brandee T. Brown

Aggressiveness of HNSCC tumors depends on expression levels of cortactin, a gene in the 11q13 amplicon

11q13 amplification is a late-stage event in several cancers that is often associated with poor prognosis. Among 11q13-amplified genes, the actin assembly protein cortactin/CTTN is considered a likely candidate for direct involvement in tumor progression because of its cell motility-enhancing functions. We modulated cortactin expression in head and neck squamous cell carcinoma (HNSCC) cell lines. Cortactin expression levels directly correlated with tumor size, vascularization and cell proliferation in an orthotopic HNSCC in vivo model. In contrast, under normal in vitro culture conditions, cortactin expression levels had no effect on cell proliferation. However, cell lines in which cortactin expression was reduced by knockdown (KD) grew poorly in vitro under harsh conditions of growth factor deprivation, anchorage independence and space constraint. In contrast, overexpression of cortactin enhanced in vitro growth under the same harsh conditions. Surprisingly, defects in growth factor-independent proliferation of cortactin-KD cells were rescued by coculture with cortactin-expressing cells. As the cocultured cells are separated by permeable filters, cortactin-expressing cells must secrete growth-supporting autocrine factors to rescue the cortactin-KD cells. Overall, cortactin expression modulates multiple cellular traits that may allow survival in a tumor environment, suggesting that the frequent overexpression of cortactin in tumors is not an epiphenomenon but rather promotes tumor aggressiveness.

Network analysis of the focal adhesion to invadopodia transition identifies a PI3K-PKCα invasive signaling axis.

The activity of the lipid kinase PI3K governs whether the protein kinase PKCα promotes invasive behavior of cancer cells. Becoming Invasive Invasive and metastatic cancer cells form cellular protrusions called invadopodia that can degrade the extracellular matrix. Hoshino et al. integrated data from head and neck carcinomas with network analysis of invadopodia and focal adhesions, cellular structures that contain many of the same components as invadopodia but have decreased ability to degrade the extracellular matrix. They identified phosphatidylinositol 3-kinase (PI3K) and protein kinase C α (PKCα) as key determinants in the formation of invadopodia. The formation of invadopodia was enhanced by PKCα in cells with wild-type PI3K but was inhibited by PKCα in cells with enhanced PI3K activity (due to expression of components of the PI3K pathway with cancer-associated mutations). These results suggested that PKCα participated in a negative feedback loop that limited the activity of PI3K and thus cellular invasiveness, which the authors confirmed. The combination of high PI3K activity with low PKCα activity correlated with increased number of invadopodia in cell lines derived from head and neck carcinoma, breast cancer, or melanoma. This PI3K-high and PKCα-low signaling state may be useful as a biomarker for cancer aggressiveness. In cancer, deregulated signaling can produce an invasive cellular phenotype. We modeled the invasive transition as a theoretical switch between two cytoskeletal structures: focal adhesions and extracellular matrix–degrading invadopodia. We constructed molecular interaction networks of each structure and identified upstream regulatory hubs through computational analyses. We compared these regulatory hubs to the status of signaling components from head and neck carcinomas, which led us to analyze phosphatidylinositol 3-kinase (PI3K) and protein kinase C α (PKCα). Consistent with previous studies, PI3K activity promoted both the formation and the activity of invadopodia. We found that PI3K induction of invadopodia was increased by overexpression of SH2 (Src homology 2) domain–containing inositol 5′-phosphatase 2 (SHIP2), which converts the phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] that is produced by PI3K activity to phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], which is believed to promote invadopodia formation. Knockdown of PKCα had divergent effects on invadopodia formation, depending on the status of PI3K. Loss of PKCα inhibited invadopodia formation in cells with wild-type PI3K pathway status. Conversely, in cells with constitutively active PI3K (through activating PI3K mutants or lacking the endogenous opposing enzyme PTEN), PKCα knockdown increased invadopodia formation. Mechanistic studies revealed a negative feedback loop from PKCα that dampened PI3K activity and invasive behavior in cells with genetic hyperactivation of the PI3K pathway. These studies demonstrated the potential of network modeling as a discovery tool and identified PI3K and PKCα as interacting regulators of invasive behavior.

Downregulation of SMG-1 in HPV-Positive Head and Neck Squamous Cell Carcinoma Due to Promoter Hypermethylation Correlates with Improved Survival

Purpose: Human papillomavirus (HPV) is linked with a subset of head and neck squamous cell carcinomas (HNSCC). HPV-positive HNSCCs show a better prognosis than HPV-negative HNSCCs, which may be explained by sensitivity of the HPV-positive HNSCCs to ionizing radiation (IR). Although the molecular mechanism behind sensitivity to IR in HPV-positive HNSCCs is unresolved, DNA damage response (DDR) might be a significant determinant of IR sensitivity. An important player in the DDR, SMG-1 (suppressor with morphogenetic effect on genitalia), is a potential tumor suppressor and may therefore be deregulated in cancer. No studies have yet been conducted linking defects in SMG-1 expression with cancer. We investigated whether deregulation of SMG-1 could be responsible for defects in the DDR in oropharyngeal HNSCC. Experimental Design: Expression and promoter methylation status of SMG-1 were investigated in HNSCCs. To identify a functional link between HPV infection and SMG-1, we transfected the HPV-negative cells with an E6/E7 expression construct. SMG-1 short hairpin RNAs were expressed in HPV-negative cells to estimate survival upon IR. Results: Forced E6/E7 expression in HPV-negative cells resulted in SMG-1 promoter hypermethylation and decreased SMG-1 expression. Due to promoter hypermethylation, HPV-positive HNSCC cells and tumors express SMG-1 at lower levels than HPV-negative SCCs. Depletion of SMG-1 in HPV-negative HNSCC cells resulted in increased radiation sensitivity, whereas SMG-1 overexpression protected HPV-positive tumor cells from irradiation. Conclusions: Levels of SMG-1 expression negatively correlated with HPV status in cancer cell lines and tumors. Diminished SMG-1 expression may contribute to the enhanced response to therapy exhibited by HPV-positive HNSCCs. Clin Cancer Res; 18(5); 1257–67. ©2012 AACR.

TrkC signaling is activated in adenoid cystic carcinoma and requires NT-3 to stimulate invasive behavior

Treatment options for adenoid cystic carcinoma (ACC) of the salivary gland, a slowly growing tumor with propensity for neuroinvasion and late recurrence, are limited to surgery and radiotherapy. Based on expression analysis performed on clinical specimens of salivary cancers, we identified in ACC expression of the neurotrophin-3 receptor TrkC/NTRK3, neural crest marker SOX10, and other neurologic genes. Here, we characterize TrkC as a novel ACC marker, which was highly expressed in 17 out of 18 ACC primary-tumor specimens, but not in mucoepidermoid salivary carcinomas or head and neck squamous cell carcinoma. Expression of the TrkC ligand NT-3 and Tyr-phosphorylation of TrkC detected in our study suggested the existence of an autocrine signaling loop in ACC with potential therapeutic significance. NT-3 stimulation of U2OS cells with ectopic TrkC expression triggered TrkC phosphorylation and resulted in Ras, Erk 1/2 and Akt activation, as well as VEGFR1 phosphorylation. Without NT-3, TrkC remained unphosphorylated, stimulated accumulation of phospho-p53 and had opposite effects on p-Akt and p-Erk 1/2. NT-3 promoted motility, migration, invasion, soft-agar colony growth and cytoskeleton restructuring in TrkC-expressing U2OS cells. Immunohistochemical analysis demonstrated that TrkC-positive ACC specimens also show high expression of Bcl2, a Trk target regulated via Erk 1/2, in agreement with activation of the TrkC pathway in real tumors. In normal salivary gland tissue, both TrkC and Bcl2 were expressed in myoepithelial cells, suggesting a principal role for this cell lineage in the ACC origin and progression. Sub-micromolar concentrations of a novel potent Trk inhibitor AZD7451 completely blocked TrkC activation and associated tumorigenic behaviors. Pre-clinical studies on ACC tumors engrafted in mice showed efficacy and low toxicity of AZD7451, validating our in vitro data and stimulating more research into its clinical application. In summary, we describe in ACC a previously unrecognized pro-survival neurotrophin signaling pathway and link it with cancer progression.

Reverse Phase Protein Array profiling of oropharyngeal cancer and significance of PIK3CA mutations in HPV-associated head and neck cancer

PURPOSE Human papilloma virus (HPV)-associated (HPV+) oropharyngeal squamous cell carcinomas (OPSCC) have different molecular and biologic characteristics and clinical behavior compared with HPV-negative (HPV-) OPSCC. PIK3CA mutations are more common in HPV(+) OPSCC. To define molecular differences and tumor subsets, protein expression and phosphorylation were compared between HPV(+) and HPV(-) OPSCC and between tumors with and without PIK3CA mutations. EXPERIMENTAL DESIGN Expression of 137 total and phosphorylated proteins was evaluated by reverse-phase protein array in 29 HPV(+) and 13 HPV(-) prospectively collected OPSCCs. Forty-seven OPSCCs were tested for hotspot-activating mutations in PIK3CA and AKT. Activation of PIK3CA downstream targets and sensitivity to pathway inhibitors were determined in HPV(+) head and neck cancer cells overexpressing wild-type or mutant PIK3CA. RESULTS Analyses revealed 41 differentially expressed proteins between HPV(+) and HPV(-) OPSCC categorized into functional groups: DNA repair, cell cycle, apoptosis, phosphoinositide 3-kinase (PI3K)/AKT/mTOR, and receptor kinase pathways. All queried DNA repair proteins were significantly upregulated in HPV(+) samples. A total of 8 of 33 HPV(+) and 0 of 14 HPV(-) tumors contained activating PIK3CA mutations. Despite all activating PIK3CA mutations occurring in HPV(+) samples, HPV(+) tumors had lower mean levels of activated AKT and downstream AKT target phosphorylation. Ectopic expression of mutant PIK3CA in HPV(+) cells increased mTOR, but not AKT activity. HPV E6/E7 overexpression inhibited AKT phosphorylation in HPV-negative cells. Mutant PIK3CA overexpressing cells were more sensitive to a dual PI3K/mTOR inhibitor compared with an AKT inhibitor. CONCLUSIONS Protein expression analyses suggest that HPV(+) and HPV(-) OPSCC differentially activate DNA repair, cell cycle, apoptosis, PI3K/AKT/mTOR, and receptor kinase pathways. PIK3CA mutations are more common in HPV(+) OPSCC and are associated with activation of mTOR, but not AKT. These data suggest that inhibitors for mTOR may have activity against HPV(+) PIK3CA mutant oropharyngeal cancers.

Proteomic analysis of oropharyngeal carcinomas reveals novel HPV-associated biological pathways.

Oropharyngeal carcinoma (OPC) can be classified into two equally prevalent subtypes depending on the presence of human papillomavirus (HPV). Patients with HPV‐positive (HPV+) OPC represent a unique cohort with a distinct tumor biology and clinical behavior compared to HPV‐negative (HPV−) OPC. Genetic studies have demonstrated chromosomal and gene expression changes associated with distinct subclasses of OPC; however, the proteomic consequences of HPV infection are not known. We analyzed sets of ten HPV+ and ten HPV− OPCs and ten normal adult oral epithelia using a standardized global proteomic analysis platform. This analysis yielded a total of 2,653 confidently identified proteins from which we chose 31 proteins on the basis of expression differences between HPV+, HPV− and normal epithelium for targeted protein quantitation. Analysis of differentially expressed proteins by HPV status revealed enrichment of proteins involved in epithelial cell development, keratinization and extracellular matrix organization in HPV− OPC, whereas enrichment of proteins in DNA initiation and replication and cell cycle control was found for HPV+ OPC. Enrichment analysis for transcription factor targets identified transcription factors E2F1 and E2F4 to be highly expressed in HPV+ OPC. We also found high expression of argininosuccinate synthase 1 in HPV+ OPC, suggesting that HPV+ OPC is more dependent on conditionally essential amino acid, arginine, and this was confirmed on a OPC‐specific tissue microarray. These identified proteomic changes reveal novel driving molecular pathways for HPV+ and HPV− OPCs that may be pertinent in therapeutic strategies and outcomes of OPC.

Human-in-mouse modeling of primary head and neck squamous cell carcinoma.

To develop a reliable modeling system for head and neck squamous cell carcinoma (HNSCC).

LIM-Only Protein 4 (LMO4) and LIM Domain Binding Protein 1 (LDB1) Promote Growth and Metastasis of Human Head and Neck Cancer (LMO4 and LDB1 in Head and Neck Cancer).

Squamous cell carcinoma of the head and neck (HNSCC) accounts for more than 300,000 deaths worldwide per year as a consequence of tumor cell invasion of adjacent structures or metastasis. LIM-only protein 4 (LMO4) and LIM-domain binding protein 1 (LDB1), two directly interacting transcriptional adaptors that have important roles in normal epithelial cell differentiation, have been associated with increased metastasis, decreased differentiation, and shortened survival in carcinoma of the breast. Here, we implicate two LDB1-binding proteins, single-stranded binding protein 2 (SSBP2) and 3 (SSBP3), in controlling LMO4 and LDB1 protein abundance in HNSCC and in regulating specific tumor cell functions in this disease. First, we found that the relative abundance of LMO4, LDB1, and the two SSBPs correlated very significantly in a panel of human HNSCC cell lines. Second, expression of these proteins in tumor primaries and lymph nodes involved by metastasis were concordant in 3 of 3 sets of tissue. Third, using a Matrigel invasion and organotypic reconstruct assay, CRISPR/Cas9-mediated deletion of LDB1 in the VU-SCC-1729 cell line, which is highly invasive of basement membrane and cellular monolayers, reduced tumor cell invasiveness and migration, as well as proliferation on tissue culture plastic. Finally, inactivation of the LDB1 gene in these cells decreased growth and vascularization of xenografted human tumor cells in vivo. These data show that LMO4, LDB1, and SSBP2 and/or SSBP3 regulate metastasis, proliferation, and angiogenesis in HNSCC and provide the first evidence that SSBPs control LMO4 and LDB1 protein abundance in a cancer context.

Viable biobanking of primary head and neck squamous cell carcinoma

To determine the feasibility of viable storage of head and neck squamous cell carcinoma (HNSCC) for regrowth of cells in culture.