Integration of high-risk human papillomavirus into cellular cancer-related genes in head and neck cancer cell lines.

BACKGROUND: Human papillomavirus (HPV)-positive oropharyngeal cancer is generally associated with excellent response to therapy, but some HPV-positive tumors progress despite aggressive therapy. The purpose of this study was to evaluate viral oncogene expression and viral integration sites in HPV16- and HPV18-positive squamous cell carcinoma lines. METHODS: E6/E7 alternate transcripts were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Detection of integrated papillomavirus sequences (DIPS-PCR) and sequencing identified viral insertion sites and affected host genes. Cellular gene expression was assessed across viral integration sites. RESULTS: All HPV-positive cell lines expressed alternate HPVE6/E7 splicing indicative of active viral oncogenesis. HPV integration occurred within cancer-related genes TP63, DCC, JAK1, TERT, ATR, ETV6, PGR, PTPRN2, and TMEM237 in 8 head and neck squamous cell carcinoma (HNSCC) lines but UM-SCC-105 and UM-GCC-1 had only intergenic integration. CONCLUSION: HPV integration into cancer-related genes occurred in 7 of 9 HPV-positive cell lines and of these 6 were from tumors that progressed. HPV integration into cancer-related genes may be a secondary carcinogenic driver in HPV-driven tumors. © 2017 Wiley Periodicals, Inc. Head Neck 39: 840-852, 2017.

STAT3 Inhibition as a Therapeutic Strategy for Chordoma.

Objective Signal transducer and activator of transcription (STAT) proteins regulate key cellular fate decisions including proliferation and apoptosis. STAT3 overexpression induces tumor growth in multiple neoplasms. STAT3 is constitutively activated in chordoma, a tumor with a high recurrence rate despite maximal surgical and radiation treatment. We hypothesized that a novel small molecule inhibitor of STAT3 (FLLL32) would induce significant cytotoxicity in sacral and clival chordoma cells. Methods Sacral (UCh1) and clival (UM-CHOR-1) chordoma cell lines were grown in culture (the latter derived from primary tumor explants). FLLL32 dosing parameters were optimized using cell viability assays. Antitumor potential of FLLL32 was assessed using clonal proliferation assays. Potential mechanisms underlying observed cytotoxicity were examined using immunofluorescence assays. Results FLLL32 induced significant cytotoxicity in UCh1 and UM-CHOR-1 chordoma cells, essentially eliminating all viable cells, correlating with observed downregulation in activated, phosphorylated STAT3 upon administration of FLLL32. Mechanisms underlying the observed cytotoxicity included increased apoptosis and reduced cellular proliferation through inhibition of mitosis. Conclusion As a monotherapy, FLLL32 induces potent tumor kill in vitro in chordoma cell lines derived from skull base and sacrum. This effect is mediated through inhibition of STAT3 phosphorylation, increased susceptibility to apoptosis, and suppression of cell proliferation.

Cancer stem cells: mediators of tumorigenesis and metastasis in head and neck squamous cell carcinoma.

BACKGROUND: Cancer stem cells (CSCs) represent a subpopulation of cells responsible for tumor growth. Their role in head and neck squamous cell carcinoma (HNSCC) tumorigenesis and metastasis remains uncertain. METHODS: Wound healing and an orthotopic animal model were used to study cells expressing the CSC phenotype (CD44(high) and aldehyde dehydrogenase [ALDH](+)) and assess mobility, tumorigenesis, and metastasis. A prospective collection of 40 patient-derived primary HNSCC specimens were analyzed for CSC-proportion compared to clinical variables. RESULTS: CSCs exhibited significantly faster wound closure and greater tumorigenesis and regional metastasis in vivo than non-CSCs. In primary patient tumors, size and advanced stage were correlated with elevated proportion of CSCs, however, not with survival. CONCLUSION: HNSCC stem cells mediate tumorigenesis and regional metastasis in vivo. In primary patient tumors, CSC-proportion was associated with tumor size and stage, but not with metastatic spread or survival. CSC burden alone may only represent a minor variable in understanding CSCs and metastasis.

UM-SCC-103: a unique tongue cancer cell line that recapitulates the tumorigenic stem cell population of the primary tumor.

OBJECTIVE: A new head and neck cancer cell line was developed from a highly aggressive HNSCC of the oral cavity diagnosed in a 26-year-old pregnant woman. METHODS: Cells from the primary tumor were passaged in culture and genotyped as a unique cell line. The resultant cell line was assessed for its ability to replicate the primary tumor. RESULTS: The primary tumor and cell line contained 19.03% and 19.62% CD44(high) cells, respectively. CD44(high) cancer stem cells from UM-SCC-103 formed tumors after flank injections in mice that reconstituted the heterogeneity of the primary tumor. CD44 staining and histology in the primary tumor and tumors grown in vivo from the cell line were similar. CD44(high) cells from the primary tumor resulted in lung colony formation in 2 out of 2 tail vein injections in mice, whereas CD44(low) cells did not. Similarly, CD44(high) cells from UM-SCC-103 formed lung tumors in 2 out of 4 mice, whereas CD44(low) cells did not. CONCLUSION: The similarity in marker expression and tumorigenic behavior between the primary tumor and the resulting cell line strongly suggests that the cell line resembles the primary tumor that it was derived from and provides an important new research tool for the study of head and neck carcinomas in young patients.

Generation of a novel dendritic-cell vaccine using melanoma and squamous cancer stem cells.

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDH(high) CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c. tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFNγ and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Head and neck cancer stem cells: the effect of HPV--an in vitro and mouse study.

OBJECTIVES: To determine if the behavior of cancer stem cells (CSCs) is affected by human papillomavirus (HPV) status. STUDY DESIGN: An in vitro and in vivo analysis of HPV and CSCs. SETTING: University laboratory. SUBJECTS AND METHODS: We isolated CSCs from HPV-positive and HPV-negative cell lines. Two HPV-negative cell lines underwent lentiviral transduction of E6/E7. Chemoresistence was determined using colony formation assays. Native HPV-positive and HPV E6/E7-transduced cells were compared for lung colonization after tail vein injection in NOD/SCID mice. RESULTS: The proportion of CSC is not significantly different in HPV-positive or HPV-negative head and neck squamous cell carcinoma (HNSCC) cell lines. The HNSCC CSCs are more resistant to cisplatin than the non-CSCs, but there were no significant differences between HPV-positive and HPV-negative cells. The HPV-negative cancer cells yielded low colony formation after cell sorting. After transduction with HPV E6/E7, increased colony formation was observed in both CSCs and non-CSCs. Results from tail vein injections yielded no differences in development of lung colonies between HPV E6/E7-transduced cells and nontransduced cells. CONCLUSIONS: Human papillomavirus status does not correlate with the proportion of CSCs present in HNSCC. The HPV-positive cells and those transduced with HPV E6/E7 have a greater clonogenicity than HPV-negative cells. The HNSCC CSCs are more resistant to cisplatin than non-CSCs. This suggests that common chemotherapeutic agents may shrink tumor bulk by eliminating non-CSCs, whereas CSCs have mechanisms that facilitate evasion of cell death. Human papillomavirus status does not affect CSC response to cisplatin therapy, suggesting that other factors explain the better outcomes for patients with HPV-positive cancer.

In vitro evaluation of sialyl Lewis X relationship with head and neck cancer stem cells.

OBJECTIVE: To evaluate in vitro the potential links between sialyl Lewis X (sLeX) and cancer stem cells (CSC) in head and neck squamous cell carcinoma (HNSCC). HNSCC is an aggressive malignancy with high mortality mainly due to metastasis. CSC have emerged as important players in HNSCC metastasis. sLeX is a tetrasaccharide carbohydrate known to play a key role in metastatic dissemination by promoting binding of the tumor cells to the endothelium. STUDY DESIGN: Experimental, in vitro. SETTING: Laboratory of Head and Neck Cancer Metastasis, University of Michigan. SUBJECTS AND METHODS: A panel of stage- and anatomic-site specific primary and metastatic HNSCC cell lines was assessed by flow cytometry to quantify sLeX relative expression levels. Serum-free conditioned media from the same HNSCC lines was collected over a time course of 72 hours and assessed by Western blot for secreted sLeX expression. Representative HNSCC cell lines were cultured as floating orospheres (condition that enhance CSC growth) or under normal adherent conditions and characterized by flow cytometry for CSC markers (CD44, aldehyde dehydrogenase [ALDH]) comparatively with sLeX expression. RESULTS: sLeX is predominantly expressed in carcinomas originating from the oral cavity. Secreted sLeX is also found to be high in oral carcinomas and increased over the analyzed time course. Floating orospheres were strongly positive for CD44 and ALDH, confirming CSC enrichment of the orospheres. Tumor cells grown as orospheres are 95% to 100% positive for sLeX compared to 10% to 40% of adherent counterpart. CONCLUSION: These studies provide the first evidence of sLeX relationship with CSC in HNSCC.

UM-SCC-104: a new human papillomavirus-16-positive cancer stem cell-containing head and neck squamous cell carcinoma cell line.

BACKGROUND: Few human papillomavirus (HPV)(+) head and neck squamous cell carcinoma (HNSCC) cell lines exist. We established University of Michigan-squamous cell carcinoma-104 (UM-SCC-104), a new HPV(+) HNSCC cell line from a recurrent oral cavity tumor, and characterized it for the presence of cancer stem cells (CSCs). METHODS: Tumor cells were tested for biomarker expression by immunohistology, and the presence of HPV was assessed by several methods. RESULTS: UM-SCC-104 has a unique genotype, contains HPV-16, and expresses E6/E7. Inoculation of aldehyde dehydrogenase (ALDH)(+) and ALDH(-) cells in an immunocompromised mouse resulted in tumor growth from the ALDH(+) cells after 6 weeks that recapitulated the histology of the primary, whereas ALDH(-) cells did not produce tumors. CONCLUSION: UM-SCC-104, a new HPV-16, CSC-containing HNSCC cell line will aid in studying recurrent HPV(+) tumors. The aggressive nature of this tumor is consistent with high uniform expression of epidermal growth factor receptor (EGFR) and a functionally significant proportion of ALDH(+) CSCs.

Head and neck cancer stem cells: the side population.

OBJECTIVES/HYPOTHESIS: The cancer stem cell (CSC) theory concludes that a subpopulation of cancer cells, the cancer stem cells, can self-renew and are responsible for tumor growth. Previous studies have identified cells able to efflux Hoechst 33342 dye as the side population (SP). SP cells and CSCs share many characteristics, suggesting the SP isolated from malignant tumors contains CSCs. STUDY DESIGN: Experimental Study. METHODS: The SP was isolated from a head and neck cancer cell line and analyzed for CSC-like characteristics. RESULTS: The SP demonstrated the ability to reproduce both SP and non-side population (NSP) cells from as few as one cell. The SP had lower expression of active ß-catenin and more resistance to 5-fluorouracil; the SP also demonstrated greater expression of Bmi-1 (4.3-fold) and ABCG2 (1.4-fold). SP cells were able to produce tumors in an animal model, whereas NSP were not. SPs were identified in two primary human tumors. CONCLUSIONS: This work adds to the evidence that the SP in head and neck cancer represents cells with CSC properties and provides a method by which CSCs can be isolated and studied.

Metastatic potential of cancer stem cells in head and neck squamous cell carcinoma.

OBJECTIVE: to design in vitro and in vivo models of metastasis to study the behavior of cancer stem cells (CSCs) in head and neck squamous cell carcinoma (HNSCC). DESIGN: cells were sorted for CD44 expression using flow cytometry. Sorted cells were used in an in vitro invasion assay. For in vivo studies, CSCs and non-CSCs were injected into the tail veins of mice, and lungs were either harvested or imaged to evaluate for lesions. RESULTS: in vitro, CD44(high) cells were more motile but not more invasive than CD44(low) cells. In vivo, 8 of 17 mice injected with CD44(high) cells and 0 of 17 mice injected with CD44(low) cells developed lung lesions. Two of the lesions arose from CSCs from a primary tumor and 6 from CSCs from HNSCC cell lines. CONCLUSIONS: in vitro, CSCs do not have an increased ability to invade through basement membrane, but they migrate more efficiently through a porous barrier. In contrast, CSCs efficiently formed lung lesions in vivo, whereas non-CSCs did not give rise to any distant disease. This phenomenon could be due to the enhanced migratory capacity of CSCs, which may be more important than basement membrane degradation in vivo.