Sanne R. Martens-de Kemp

Functional Genetic Screens Identify Genes Essential for Tumor Cell Survival in Head and Neck and Lung Cancer

Purpose: Despite continuous improvement of treatment regimes, the mortality rates for non–small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) remain disappointingly high and novel anticancer agents are urgently awaited. Experimental Design: We combined the data from genome-wide siRNA screens on tumor cell lethality in a lung and a head and neck cancer cell line. Results: We identified 71 target genes that seem essential for the survival of both cancer types. We identified a cluster of 20 genes that play an important role during G2–M phase transition, underlining the importance of this cell-cycle checkpoint for tumor cell survival. Five genes from this cluster (CKAP5, KPNB1, RAN, TPX2, and KIF11) were evaluated in more detail and have been shown to be essential for tumor cell survival in both tumor types, but most particularly in HNSCC. Phenotypes that were observed following siRNA-mediated knockdown of KIF11 (kinesin family member 11) were reproduced by inhibition of KIF11 using the small-molecule inhibitor ispinesib (SB-715992). We showed that ispinesib induces a G2 arrest, causes aberrant chromosome segregation, and induces cell death in HNSCC in vitro, whereas primary keratinocytes are less sensitive. Furthermore, growth of HNSCC cells engrafted in immunodeficient mice was significantly inhibited after ispinesib treatment. Conclusion: This study identified a wide array of druggable genes for both lung and head and neck cancer. In particular, multiple genes involved in the G2–M checkpoint were shown to be essential for tumor cell survival, indicating their potential as anticancer targets. Clin Cancer Res; 19(8); 1994–2003. ©2013 AACR.

Cancer stem cell enrichment marker CD98: A prognostic factor for survival in patients with human papillomavirus-positive oropharyngeal cancer

PURPOSE Several hypotheses have been proposed to explain the relatively good prognosis of patients with a human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) and one of these is a higher sensitivity to (chemo)radiation. Previous studies have suggested that treatment failure in OPSCC patients is caused by resistance of cancer stem cells (CSCs). The purpose of this study was to evaluate the association between the number of CSCs and prognosis in HPV-positive OPSCC patients. EXPERIMENTAL DESIGN All OPSCC patients (n=711) treated between 2000 and 2006 in two Dutch university hospitals were included. Presence of HPV in a tumour tissue specimen was tested by p16-immunostaining followed by HPV DNA GP5+/6+polymerase chain reaction (PCR). The presence and intensity of tumour CSC markers CD44 and CD98 were determined by immunohistochemistry and semiquantitative scoring was performed. Overall survival (OS) and progression-free survival (PFS) rates were compared between patients with low and high CD44/CD98 expression in relation to HPV status. RESULTS HPV-positive tumours showed a lower percentage of cells with CD44 and CD98 expression than HPV-negative tumours (p<0.001, χ(2)-test). Within the group of patients with HPV-positive OPSCC, a high percentage of CD98-positive tumour cells was associated with a significantly worse 5-year OS and PFS (OS: 36.4% and PFS: 27.3%) compared to patients with a low percentage of CD98-positive cells (OS: 71.9% and PFS: 70.5%, respectively) (p<0.001). CONCLUSIONS HPV-positive OPSCCs harbour fewer cells expressing the CSC enrichment markers CD44 and CD98. Furthermore, OS and PFS were significantly worse for patients with HPV-positive OPSCC with a high percentage of CD98-positive cells.

Treatment response of HPV-positive and HPV-negative head and neck squamous cell carcinoma cell lines

OBJECTIVES Infection with the human papillomavirus (HPV) is an important risk factor for development of head and neck squamous cell carcinoma (HNSCC). Strikingly, HPV-positive HNSCCs have a more favorable prognosis than their HPV-negative counterparts. The current study was designed to explain this favorable prognosis of HPV-positive HNSCC. MATERIALS AND METHODS This was performed by investigating the response of four HPV-positive and fourteen HPV-negative HNSCC cell lines to cisplatin, cetuximab and radiation. RESULTS Analysis of the responses of this cell line panel indicated that HPV-positive cells are more resistant to cisplatin treatment than the HPV-negative HNSCCs, whereas the response to radiation and cetuximab did not differ. CONCLUSIONS The current study suggests that the favorable prognosis for patients with HPV-positive HNSCC does not seem to be related to an intrinsic sensitivity of these tumor cells to chemotherapy or radiation in vitro.

DNA-Bound Platinum Is the Major Determinant of Cisplatin Sensitivity in Head and Neck Squamous Carcinoma Cells

Purpose The combination of systemic cisplatin with local and regional radiotherapy as primary treatment of head and neck squamous cell carcinoma (HNSCC) leads to cure in approximately half of the patients. The addition of cisplatin has significant effects on outcome, but despite extensive research the mechanism underlying cisplatin response is still not well understood. Methods We examined 19 HNSCC cell lines with variable cisplatin sensitivity. We determined the TP53 mutational status of each cell line and investigated the expression levels of 11 potentially relevant genes by quantitative real-time PCR. In addition, we measured cisplatin accumulation and retention, as well as the level of platinum-DNA adducts. Results We found that the IC50 value was significantly correlated with the platinum-DNA adduct levels that accumulated during four hours of cisplatin incubation (p = 0.002). We could not find a significant correlation between cisplatin sensitivity and any of the other parameters tested, including the expression levels of established cisplatin influx and efflux transporters. Furthermore, adduct accumulation did not correlate with mRNA expression of the investigated influx pumps (CTR1 and OCT3) nor with that of the examined DNA repair genes (ATR, ATM, BRCA1, BRCA2 and ERCC1). Conclusion Our findings suggest that the cisplatin-DNA adduct level is the most important determinant of cisplatin sensitivity in HNSCC cells. Imaging with radio-labeled cisplatin might have major associations with outcome.

CD98 marks a subpopulation of head and neck squamous cell carcinoma cells with stem cell properties

Patients with advanced head and neck squamous cell carcinomas (HNSCCs) are often treated with concomitant chemotherapy and radiotherapy, but only 50% is cured. A possible explanation for treatment failure is therapy resistance of the cancer stem cells (CSCs). The application of compounds specifically targeting these CSCs, in addition to routinely used therapeutics, would likely improve clinical outcome. We demonstrate that the previously described monoclonal antibody K984 recognizes the CD98 cell surface protein, which is specifically expressed by cells forming the squamous basal cell layer, the region where the squamous stem cells reside. Moreover, CD98 is highly resistant to the proteolytic enzymes required for CSC enrichment procedures. We show that CD98(high) cells, in contrast to CD98(low) cells, are able to generate tumors in immunodeficient mice, indicating that CD98(high) cells have stem cell characteristics. Furthermore, the CD98(high) subpopulation expresses high levels of cell cycle control and DNA repair genes, while the CD98(low) fraction shows expression patterns that represent the more differentiated cells forming the bulk of the tumor. CD98 is a promising CSC enrichment marker in HNSCC. Our data support the CSC concept in head and neck cancer and the potential relevance of these cells for treatment outcome.

CRK7 modifies the MAPK pathway and influences the response to endocrine therapy

Endocrine therapies, which inhibit estrogen receptor (ER)alpha signaling, are the most common and effective treatment for ERalpha-positive breast cancer. However, the use of these agents is limited by the frequent development of resistance. The cyclin-dependent kinase family member CRK7 (aka CRKRS) was identified from an RNA interference screen for modifiers of tamoxifen sensitivity. Here, we demonstrate that silencing of CRK7 not only causes resistance to tamoxifen but also leads to resistance to additional endocrine therapies including ICI 182780 and estrogen deprivation, a model of aromatase inhibition. We show that CRK7 silencing activates the mitogen-activated protein kinase (MAPK)-signaling pathway, which causes a loss of ER dependence, resulting in endocrine therapy resistance. This study identifies a novel role for CRK7 in MAPK regulation and resistance to estrogen signaling inhibitors.

The FA/BRCA pathway identified as the major predictor of cisplatin response in head and neck cancer by functional genomics.

Patients with advanced stage head and neck squamous cell carcinoma (HNSCC) are often treated with cisplatin-containing chemoradiation protocols. Although cisplatin is an effective radiation sensitizer, it causes severe toxicity and not all patients benefit from the combination treatment. HNSCCs expectedly not responding to cisplatin may better be treated with surgery and postoperative radiation or cetuximab and radiation, but biomarkers to personalize chemoradiotherapy are not available. We performed an unbiased genome-wide functional genetic screen in vitro to identify genes that influence the response to cisplatin in HNSCC cells. By siRNA-mediated knockdown, we identified the Fanconi anemia/BRCA pathway as the predominant pathway for cisplatin response in HNSCC cells. We also identified the involvement of the SHFM1 gene in the process of DNA cross-link repair. Furthermore, expression profiles based on these genes predict the prognosis of radiation- and chemoradiation-treated head and neck cancer patients. This genome-wide functional analysis designated the genes that are important in the response of HNSCC to cisplatin and may guide further biomarker validation. Cisplatin imaging as well as biomarkers that indicate the activity of the Fanconi anemia/BRCA pathway in the tumors are the prime candidates. Mol Cancer Ther; 16(3); 540–50. ©2016 AACR.

Targeting PLK1 as a novel chemopreventive approach to eradicate preneoplastic mucosal changes in the head and neck

Head and neck squamous cell carcinomas (HNSCC) and local relapses thereof develop in preneoplastic fields in the mucosal linings of the upper aerodigestive tract. These fields are characterized by tumor-associated genetic changes, are frequently dysplastic and occasionally macroscopically visible. Currently, no adequate treatment options exist to prevent tumor development. Array-based screening with a panel of tumor-lethal small interfering RNAs (siRNAs) identified Polo-like kinase 1 (PLK1) as essential for survival of preneoplastic cells. Inhibition of PLK1 caused cell death of preneoplastic and HNSCC cells, while primary cells were hardly affected. Both siRNAs and small molecule inhibitors caused a strong G2/M cell cycle arrest accompanied by formation of monopolar spindles. In a xenografted mouse model PLK1 caused a significant tumor growth delay and cures, while chemoradiation had no effect. Thus, PLK1 seems to be a promising target for chemopreventive treatment of preneoplastic cells, and could be applied to prevent HNSCC and local relapses.Head and neck squamous cell carcinomas (HNSCC) and local relapses thereof develop in preneoplastic fields in the mucosal linings of the upper aerodigestive tract. These fields are characterized by tumor-associated genetic changes, are frequently dysplastic and occasionally macroscopically visible. Currently, no adequate treatment options exist to prevent tumor development. Array-based screening with a panel of tumor-lethal small interfering RNAs (siRNAs) identified Polo-like kinase 1 (PLK1) as essential for survival of preneoplastic cells. Inhibition of PLK1 caused cell death of preneoplastic and HNSCC cells, while primary cells were hardly affected. Both siRNAs and small molecule inhibitors caused a strong G2/M cell cycle arrest accompanied by formation of monopolar spindles. In a xenografted mouse model PLK1 caused a significant tumor growth delay and cures, while chemoradiation had no effect. Thus, PLK1 seems to be a promising target for chemopreventive treatment of preneoplastic cells, and could be applied to prevent HNSCC and local relapses.

Abstract 4638: Targeted therapy of precancerous fields in the mucosal linings of the head and neck

Primary head and neck squamous cell carcinomas (HNSCC) and local relapses develop in preneoplastic fields in the mucosal linings of the upper aerodigestive tract. These fields are characterized by tumor-associated genetic changes, show frequently dysplastic features and are occasionally macroscopically visible as lesions. Currently, there are no adequate treatment options for these fields to prevent tumor development. To develop a targeted therapeutic approach we established four unique preneoplastic cell lines, derived from fields adjacent to HNSCCs. Next, we performed a screen with a selective panel of small interfering RNAs (siRNAs) that are lethal for HNSCC cells, to identify genes essential for survival of preneoplastic cells and not for normal cells. One of these selective essential genes was Polo-like kinase 1 (PLK1). Inhibition of PLK1, both with siRNAs as well as small molecule inhibitors, caused cell death of preneoplastic and HNSCC tumor cells, while primary fibroblasts and keratinocytes were hardly affected. Targeting PLK1 caused a strong G2/M cell cycle arrest in preneoplastic cells accompanied by formation of monopolar spindles during mitosis. Hence, PLK1 is a promising therapeutic target for chemopreventive eradication of preneoplastic fields in patients at high risk of developing HNSCC and local relapses. Citation Format: D Vicky de Boer, Sanne R. Martens-de Kemp, Marijke Buijze, Marijke Stigter-van Walsum, Elisabeth Bloemena, C Rene Leemans, Boudewijn J.M. Braakhuis, Ruud H. Brakenhoff. Targeted therapy of precancerous fields in the mucosal linings of the head and neck. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4638.

Abstract 1750: Identification of genes involved in chemoradiation response of head and neck cancer by functional genetic screens

Introduction: Patients with advanced head and neck squamous cell carcinoma (HNSCC) are treated with cisplatin-containing chemoradiation protocols, but tumor responses can be short-lasting. For these patients surgery combined with postoperative radiotherapy is preferred over chemoradiation. Currently, there are no strong predictors of chemoradiation response that would allow to personalize treatment. We performed a functional genetic siRNA screen to identify genes that can be used to predict upfront which tumors are likely to respond to chemoradiation. Methods: A large panel of HNSCC cell lines was characterized for cisplatin sensitivity. A suitable HNSCC cell line was selected and forward transfected with a genome-wide library of short interfering RNAs (siRNAs), followed by cisplatin (IC20 and IC80) or mock treatment. Genes influencing cellular response to cisplatin were identified by comparing cell viability between the different conditions. The screen was carried out in duplicate and data were analyzed by Z-score calculation using two independent statistical methods. Results: Scrambled siRNAs and siRNAs targeting genes known to influence cell viability or cisplatin sensitivity were included as controls and gave the expected results: less than 10% toxicity and >80% knock-down. Hit lists obtained with both methods of data analysis revealed 86 siRNAs that appeared to influence cisplatin response significantly. Some of these 86 siRNAs target genes that are known to cause sensitivity to cisplatin, thereby confirming the reliability of the approach. We also obtained a list of 80 genes that influenced basic cell viability of HNSCC cells independent of cisplatin. Conclusion: By specifically knocking down all human genes in HNSCC cells, we obtained an unbiased list of at least 86 candidate genes that seem to influence the cellular response to cisplatin. Refined statistical analysis combined with bioinformatics, the exclusion of off-target effects, as well as confirmation of the hits in multiple cell lines is needed to validate these results. Validated hits will be evaluated as predictive markers in clinical samples. This research was supported by the Center for Translational Molecular Medicine (AIRFORCE project). 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 1750. doi:10.1158/1538-7445.AM2011-1750