Hedgehog pathway mutations are involved in the pathogenesis of plaque-type "trichoblastoma": A report of two cases.

We present two cases of plaque-type trichoblastoma with atypical foci. A rare variant of trichoblastoma is the plaque variant, which is characterized by poor circumscription and locally infiltrative growth pattern. These lesions mostly require multiple stages of Mohs micrographic surgery. Debate still exists whether this variant should be considered as a benign entity or as "low-grade" malignant counterpart of trichoblastoma. In this report we describe two cases of plaque-type trichoblastoma with atypical foci, which harbored somatic mutations in the Hedgehog pathway, thus should be acknowledged as intermediate malignancies. In addition, extensive molecular workup of both the trichoblastic and atypical component in sequential lesions in the same patient was performed.

Overexpression of the miR-17-92 cluster in colorectal adenoma organoids causes a carcinoma-like gene expression signature.

Gain of chromosome arm 13q is one of the most prevalent DNA copy number alterations associated with colorectal adenoma-to-carcinoma progression. The oncogenic miR-17-92 cluster, located at 13q, was found to be overexpressed in colorectal cancer and in adenomas harboring 13q gain. However, to what extent overexpression of this group of microRNAs actually drives progression to cancer remains to be resolved. Therefore, we aimed to clarify the role of miR-17-92 cluster in the progression from colorectal adenoma to carcinoma. The miR-17-92 cluster was overexpressed in human colorectal adenoma organoids without 13q gain and downstream effects on mRNA expression were investigated, along with functional consequences in vitro and in vivo. Comparison of mRNA sequencing results of organoids overexpressing miR-17-92 and cultures transduced with control vector revealed a miR-17-92 expression signature. This signature appeared to be enriched in an independent series of colorectal cancers and adenomas with 13q gain, confirming that miR-17-92 expression is associated with malignant progression. However, tumor-associated characteristics, such as increased proliferation rate, were not observed in miR-17-92 overexpressing adenoma organoids in vitro. In addition, subcutaneous injection of these organoids in immunodeficient mice was insufficient to cause tumor outgrowth. In conclusion, this study showed that miR-17-92 expression contributes to 13q gain-associated adenoma-to-carcinoma progression, however, this is insufficient to cause malignancy.

Chemopreventive targeted treatment of head and neck precancer by Wee1 inhibition.

HPV-negative head and neck squamous cell carcinomas (HNSCCs) develop in precancerous changes in the mucosal lining of the upper-aerodigestive tract. These precancerous cells contain cancer-associated genomic changes and cause primary tumors and local relapses. Therapeutic strategies to eradicate these precancerous cells are very limited. Using functional genomic screens, we identified the therapeutic vulnerabilities of premalignant mucosal cells, which are shared with fully malignant HNSCC cells. We screened 319 previously identified tumor-lethal siRNAs on a panel of cancer and precancerous cell lines as well as primary fibroblasts. In total we identified 147 tumor-essential genes including 34 druggable candidates. Of these 34, 13 were also essential in premalignant cells. We investigated the variable molecular basis of the vulnerabilities in tumor and premalignant cell lines and found indications of collateral lethality. Wee1-like kinase (WEE1) was amongst the most promising targets for both tumor and precancerous cells. All four precancerous cell lines were highly sensitive to Wee1 inhibition by Adavosertib (AZD1775), while primary keratinocytes tolerated this inhibitor. Wee1 inhibition caused induction of DNA damage during S-phase followed by mitotic failure in (pre)cancer cells. In conclusion, we uncovered Wee1 inhibition as a promising chemopreventive strategy for precancerous cells, with comparable responses as fully transformed HNSCC cells.

Molecular characterization of colorectal adenomas reveals POFUT1 as a candidate driver of tumor progression.

Removal of colorectal adenomas is an effective strategy to reduce colorectal cancer (CRC) mortality rates. However, as only a minority of adenomas progress to cancer, such strategies may lead to overtreatment. The present study aimed to characterize adenomas by in-depth molecular profiling, to obtain insights into altered biology associated with the colorectal adenoma-to-carcinoma progression. We obtained low-coverage whole genome sequencing, RNA sequencing and tandem mass spectrometry data for 30 CRCs, 30 adenomas and 18 normal adjacent colon samples. These data were used for DNA copy number aberrations profiling, differential expression, gene set enrichment and gene-dosage effect analysis. Protein expression was independently validated by immunohistochemistry on tissue microarrays and in patient-derived colorectal adenoma organoids. Stroma percentage was determined by digital image analysis of tissue sections. Twenty-four out of 30 adenomas could be unambiguously classified as high risk (n = 9) or low risk (n = 15) of progressing to cancer, based on DNA copy number profiles. Biological processes more prevalent in high-risk than low-risk adenomas were related to proliferation, tumor microenvironment and Notch, Wnt, PI3K/AKT/mTOR and Hedgehog signaling, while metabolic processes and protein secretion were enriched in low-risk adenomas. DNA copy number driven gene-dosage effect in high-risk adenomas and cancers was observed for POFUT1, RPRD1B and EIF6. Increased POFUT1 expression in high-risk adenomas was validated in tissue samples and organoids. High POFUT1 expression was also associated with Notch signaling enrichment and with decreased goblet cells differentiation. In-depth molecular characterization of colorectal adenomas revealed POFUT1 and Notch signaling as potential drivers of tumor progression.

Targeting the cell cycle in head and neck cancer by Chk1 inhibition: a novel concept of bimodal cell death.

Head and neck squamous cell carcinomas (HNSCCs) coincide with poor survival rates. The lack of driver oncogenes complicates the development of targeted treatments for HNSCC. Here, we follow-up on two previous genome-wide RNA and microRNA interference screens in HNSCC to cross-examine tumor-specific lethality by targeting ATM, ATR, CHEK1, or CHEK2. Our results uncover CHEK1 as the most promising target for HNSCC. CHEK1 expression is essential across a panel of HNSCC cell lines but redundant for growth and survival of untransformed oral keratinocytes and fibroblasts. LY2603618 (Rabusertib), which specifically targets Chk1 kinase, kills HNSCC cells effectively and specifically. Our findings show that HNSCC cells depend on Chk1-mediated signaling to progress through S-phase successfully. Chk1 inhibition coincides with stalled DNA replication, replication fork collapses, and accumulation of DNA damage. We further show that Chk1 inhibition leads to bimodal HNSCC cell killing. In the most sensitive cell lines, apoptosis is induced in S-phase, whereas more resistant cell lines manage to bypass replication-associated apoptosis, but accumulate chromosomal breaks that become lethal in subsequent mitosis. Interestingly, CDK1 expression correlates with treatment outcome. Moreover, sensitivity to Chk1 inhibition requires functional CDK1 and CDK4/6 to drive cell cycle progression, arguing against combining Chk1 inhibitors with CDK inhibitors. In contrast, Wee1 inhibitor Adavosertib progresses the cell cycle and thereby increases lethality to Chk1 inhibition in HNSCC cell lines. We conclude that Chk1 has become a key molecule in HNSCC cell cycle regulation and a very promising therapeutic target. Chk1 inhibition leads to S-phase apoptosis or death in mitosis. We provide a potential efficacy biomarker and combination therapy to follow-up in clinical setting.

Aurora kinase A (AURKA) interaction with Wnt and Ras-MAPK signalling pathways in colorectal cancer.

Hyperactivation of Wnt and Ras-MAPK signalling are common events in development of colorectal adenomas. Further progression from adenoma-to-carcinoma is frequently associated with 20q gain and overexpression of Aurora kinase A (AURKA). Interestingly, AURKA has been shown to further enhance Wnt and Ras-MAPK signalling. However, the molecular details of these interactions in driving colorectal carcinogenesis remain poorly understood. Here we first performed differential expression analysis (DEA) of AURKA knockdown in two colorectal cancer (CRC) cell lines with 20q gain and AURKA overexpression. Next, using an exact algorithm, Heinz, we computed the largest connected protein-protein interaction (PPI) network module of significantly deregulated genes in the two CRC cell lines. The DEA and the Heinz analyses suggest 20 Wnt and Ras-MAPK signalling genes being deregulated by AURKA, whereof ß-catenin and KRAS occurred in both cell lines. Finally, shortest path analysis over the PPI network revealed eight 'connecting genes' between AURKA and these Wnt and Ras-MAPK signalling genes, of which UBE2D1, DICER1, CDK6 and RACGAP1 occurred in both cell lines. This study, first, confirms that AURKA influences deregulation of Wnt and Ras-MAPK signalling genes, and second, suggests mechanisms in CRC cell lines describing these interactions.

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.

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.

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.

Identification of lethal microRNAs specific for head and neck cancer.

PURPOSE: The prognosis of head and neck squamous cell carcinomas (HNSCC) remains disappointing and the development of novel anti-cancer agents is urgently awaited. We identified by a functional genetic screen microRNAs that are selectively lethal for head and neck cancer cells but not for normal cells. We further investigated the genes targeted by these microRNAs. EXPERIMENTAL DESIGN: A retroviral expression library of human microRNAs was introduced in HNSCC cell lines and normal oropharyngeal keratinocytes to identify tumor-selective lethal microRNAs. Potential downstream gene targets of these microRNAs were identified by gene expression profiling and validated by functional assays. RESULTS: We identified six microRNAs that selectively inhibit proliferation of head and neck cancer cells. By gene expression profiling and 3'-untranslated region (UTR) luciferase reporter assays, we showed that the ataxia telangiectasia mutated (ATM) gene is a common target for at least two and likely three of these microRNAs. Specific inhibition of ATM resulted in a similar tumor-specific lethal effect, whereas the phenotype was reverted in rescue experiments. CONCLUSIONS: These six microRNAs might be developed as novel anti-cancer agents and highlight ATM as an interesting novel therapeutic target for head and neck cancer.

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.

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.

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.

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.

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.