Transient receptor potential melastatin‐4 (TRPM4) ion channel expression is upregulated in prostate cancer (PCa), contributing to increased cell proliferation, migration, adhesion, epithelial‐to‐mesenchymal transition, cell cycle shift, and alterations of intracellular Ca2+ signaling. GEO2R platform analysis of messenger RNA (mRNA) expression of ~ 6350 genes in normal and malignant prostate tissue samples from 15 PCa patients demonstrates that TRPM4 expression is upregulated sixfold and is among the most significantly upregulated genes in PCa. We find that absence of TRPM4 reduced PCa tumor spheroid size and decreased PCa tumor spheroid outgrowth. In addition, lack of TRPM4 increased cell death in PCa tumor spheroids, a phenotype that is absent in two‐dimensional (2D) cancer cell systems. Lastly, absence of TRPM4 in PCa cells reduced extravasation and metastatic burden in a preclinical zebrafish cancer model. Taken together, our findings show that TRPM4 is an attractive therapeutic target in PCa and highlights the need for future development of pharmacological tools.

Nicotine in tobacco is known to induce tumor-promoting effects and cause chemotherapy resistance through the activation of nicotinic acetylcholine receptors (nAChRs). Many studies have associated the α5 nicotinic receptor subunit (α5), and a specific polymorphism in this subunit, with (i) nicotine administration, (ii) nicotine dependence, and (iii) lung cancer. The α5 gene CHRNA5 mRNA is upregulated in several types of cancer, including lung, prostate, colorectal, and stomach cancer, and cancer severity is correlated with smoking. In this study, we investigate the contribution of α5 in the nicotine-induced cancer hallmark functions proliferation and migration, in breast, colon, and prostate cancer cells. Nine human cell lines from different origins were used to determine nAChR subunit expression levels. Then, selected breast (MCF7), colon (SW480), and prostate (DU145) cancer cell lines were used to investigate the nicotine-induced effects mediated by α5. Using pharmacological and siRNA-based experiments, we show that α5 is essential for nicotine-induced proliferation and migration. Additionally, upon downregulation of α5, nicotine-promoted expression of EMT markers and immune regulatory proteins was impaired. Moreover, the α5 polymorphism D398N (α5SNP) caused a basal increase in proliferation and migration in the DU145 cell line, and the effect was mediated through G-protein signaling. Taken together, our results indicate that nicotine-induced cancer cell proliferation and migration are mediated via α5, adding to the characterization of α5 as a putative therapeutical target.

Although 2D in vitro cancer cell cultures have been used for decades as a first line-of-research tool to investigate antitumoral drugs and treatments, their use presents many drawbacks, including the poor resemblance of such cultures to the characteristics of in vivo tumors. To mitigate these drawbacks, 3D culture models have emerged as a more representative alternative. Cancer cells cultured as 3D structures have the advantage of resembling solid tumors in their architecture and in their resistance to chemotherapeutic drugs, in part because of restrained drug penetration. Additionally, these 3D structures create a more physiological environment for the study of immune cell invasion and migration, comparable to solid tumors. In this paper, we describe a fast and cost-effective step-by-step protocol for the generation of 3D spheres using ultra-low-attachment (ULA) multiwell plates, which can be incorporated into the normal workflow of any laboratory. Using this protocol, spheroids of different human cancer cell lines can be obtained and can then be characterized on the basis of their morphology, viability, and expression of specific markers.

Introduction Despite the development of new therapeutic approaches for head and neck squamous cell carcinomas (HNSCC), this disease is still associated with high mortality and poor prognosis. Preclinical and epidemiological studies in recent years provided preliminary evidence of a potential antitumor effect of vitamin D on HNSCC cells, but data are still insufficient and further research is needed. In our study, we analyzed the effect of calcitriol on the proliferation and migration behavior of HNSCC cells in vitro.

Einleitung Die Immunonkologie hat in den zurückliegenden Jahren zunehmend auch in der Behandlung von Kopf-Hals-Karzinompatienten (HNSCC) an Bedeutung gewonnen und Eingang in die klinische Praxis der HNO-Onkologie gefunden. Dennoch zeigen viele Patienten kein oder nur ein passageres Ansprechen auf die verfügbaren Anti-PD1 Checkpointinhibitoren, so dass weiterführende Untersuchungen zur Tumor-Immunzellinteraktion erforderlich sind um das therapeutische Spektrum zu erweitern. In diesem Kontext beschäftigten wir uns in der vorgestellten Studie mit der Relevanz der Stromaseneszenz und der T-Zell-Erschöpfung auf die Prognose von HNSCC-Patienten.

Einleitung Trotz der Entwicklung neuer Therapieansätze bei Plattenepithelkarzinomen im Kopf-Hals-Bereich (HNSCC), ist diese Erkrankung nach wie vor mit einer hohen Mortalität und einer schlechten Prognose verbunden. Präklinische und epidemiologische Studien der letzten Jahre lieferten erste Hinweise auf eine potentielle antitumorale Wirkung von Vitamin D auf HNSCC-Zellen, jedoch ist die Datenlage noch nicht ausreichend und es bedarf weiterer Forschung. In unserer Studie analysierten wir in-vitro den Einfluss von Calcitriol auf das Proliferations- und Migrationsverhalten von HNSCC-Zellen.

Einleitung Vor dem Hintergrund einer wachsenden Bedeutung der Immuncheckpoint Inhibition im Behandlungsspektrum von Kopf-Hals-Tumorpatienten (HNSCC) wurde in dieser Arbeit die prognostische Relevanz Tumor-infiltrierender Immunzellen untersucht. Hierbei wurde auf RNA Sequenzierungs-Daten der TCGA-HNSC Kohorte zurückgegriffen. Zur Validierung wurden die fünf Zellpopulationen mit der höchsten prognostischen Relevanz an einem eigenen Patientenkollektiv validiert.

Introduction Due to the increasing value of immune checkpoint inhibition in the therapeutic spectrum for HNSCC patients this scientific work carves out the prognostic relevance of tumor infiltrating immune cells. In this connection, RNA sequence data of the TCGA-HNSC cohort were used. For validation we planned a verification of the five most significant cell populations (naïve B cells, follicular T-helper cells, macrophages, regulatory T cells, lymphocytes) on a proprietary patient collective.

The transient receptor potential melastatin 4 (TRPM4) ion channel is ubiquitously expressed. Dysregulation and/or functional mutations of TRPM4 lead to several diseases. Within our studies, we screened for TRPM4 inhibitors and identified small molecules that block TRPM4 in the low µM range. Furthermore, we investigated the pathophysiology of TRPM4 in cardiac conditions, immune diseases and cancer using these novel inhibitors, molecular biology techniques and functional assays.

Introduction Head and neck squamous cell carcinomas (HNSCCs) are cancers with generally poor prognosis. Outcomes have not improved in decades, with more than half of the patients presenting with lymph node metastases at the time of diagnosis. A unique subtype of HNSCC, cancer of unknown primary of the head and neck (HNCUP) is associated with a poor outcome. Increased expression of the D2-40 gene (podoplanin) has been described for several human malignancies and has been associated with increased metastatic potential of cancer cells. Methods In order to examine the role of podoplanin in lymph node metastasis of HNSCC generally and HNCUP specifically, we evaluated the prognostic impact of podoplanin expression in HNSCC- (n = 68) and HNCUP-associated lymph node metastases (n = 30). The expression of podoplanin was analyzed by immunohistochemical staining of lymph node tissue samples and correlated with clinical and histopathological data. Results We found a non-significant tendency towards a higher podoplanin expression in HNCUP compared to HNSCC lymph node metastases and a significant correlation between a high podoplanin expression and advanced node-stage classification. Podoplanin expression had no significant impact on overall survival for both groups and did not correlate with human papillomavirus tumor status. Conclusion Taken together, our results suggest that upregulation of podoplanin may be associated with a stimulation of lymphatic metastasis in head and neck cancer.

Introduction Head and neck squamous cell carcinomas (HNSCC) have been associated with a poor prognosis for decades, with more than half of the patients already showing lymph node metastases at the time of diagnosis. The CUP syndrome (Cancer of unknown primary) of the head and neck area is special form and considered with a bad prognosis. An increased expression of the D2-40 gene (podoplanin) has already been described in some human malignancies and is associated with an increased metastatic tendency. However, only limited data are available on the role of podoplanin in HNSCCs.

Various cancer types including head and neck squamous cell carcinomas (HNSCC) show a frequent amplification of chromosomal region 3q26 that encodes, among others, for the SEC62 gene. Located in the ER membrane, this translocation protein is known to play a critical role as a potential driver oncogene in cancer development. High SEC62 expression levels were observed in various cancer entities and were associated with a poor outcome and increased metastatic burden. Because of its intracellular localization the SEC62 protein is poorly accessible for therapeutic antibodies, therefore a functional SEC62 knockdown represents the most promising mechanism of a potential antineoplastic targeted therapy. By stimulating the Ca2+ efflux from the ER lumen and thereby increasing cellular stress levels, a functional inhibition of SEC62 bears the potential to limit tumor growth and metastasis formation. In this study, two potential anti-metastatic and -proliferative agents that counteract SEC62 function were investigated in functional in vitro assays by utilizing an immortalized human hypopharyngeal cancer cell line as well as a newly established orthotopic murine in vivo model. Additionally, a CRISPR/Cas9 based SEC62 knockout HNSCC cell line was generated and functionally characterized for its relevance in HNSCC cell proliferation and migration as well as sensitivity to SEC62 targeted therapy in vitro.

Introduction Immunooncology has gained importance in the treatment of head and neck cancer patients (HNSCC) over the past years and has found its way into the clinical practice of head and neck oncology. Nevertheless, many patients show no or only a transient response to anti-PD1 antibodies, so that further investigations of tumor-immune cell interaction are necessary to expand the spectrum of immunotherapy. In this context, we addressed the relevance of stroma senescence and T cell exhaustion on the prognosis of HNSCC patients in the presented study.

SEC62 oncogene located at chromosomal region 3q26 encodes for a transmembrane protein of the endoplasmic reticulum (ER) and is expressed at high levels in numerous human malignancies. SEC62 overexpression has been associated with worse prognosis and high risk for lymphatic and distant metastases in head and neck cancer, cervical cancer, hepatocellular cancer, and lung cancer. However, its role in the development and tumor biology of melanocytic lesions has not been investigated so far. An immunohistochemical study including 209 patients with melanocytic lesions (malignant melanoma (MM), n = 93; melanoma metastases (MET), n = 28; Spitz nevi (SN), n = 29; blue nevi (BN), n = 21; congenital nevi (CN), n = 38) was conducted and SEC62 expression was correlated with clinical data including patient survival and histopathological characteristics. SN showed the highest SEC62 expression levels followed by MET, MM, CN, and BN. High SEC62 expression correlated with a shorter overall and progression-free survival in MM patients. Additionally, high Sec62 levels correlated significantly with higher tumor size (T stage), the presence of tumor ulceration, and the presence of lymph node as well as distant metastases. Strikingly, SEC62 expression showed a strong correlation with Clark level. Taken together, these data demonstrate that SEC62 is a promising prognostic marker in MM and has the potential to predict biological behavior and clinical aggressiveness of melanocytic lesions.

The incidence of human papillomavirus (HPV)-related head and neck cancer (HNSCC) is rising globally, presenting challenges for optimized clinical management. To date, it remains unclear which biomarker best reflects HPV-driven carcinogenesis, a process that is associated with better therapeutic response and outcome compared to tobacco/alcohol-induced cancers. Six potential HPV surrogate biomarkers were analyzed using FFPE tissue samples from 153 HNSCC patients (n = 78 oropharyngeal cancer (OPSCC), n = 35 laryngeal cancer, n = 23 hypopharyngeal cancer, n = 17 oral cavity cancer): p16, CyclinD1, pRb, dual immunohistochemical staining of p16 and Ki67, HPV-DNA-PCR, and HPV-DNA-in situ hybridization (ISH). Biomarkers were analyzed for correlation with one another, tumor subsite, and patient survival. P16-IHC alone showed the best performance for discriminating between good (high expression) vs poor outcome (low expression; p = 0.0030) in OPSCC patients. Additionally, HPV-DNA-ISH (p = 0.0039), HPV-DNA-PCR (p = 0.0113), and p16-Ki67 dual stain (p = 0.0047) were significantly associated with prognosis in uni- and multivariable analysis for oropharyngeal cancer. In the non-OPSCC group, however, none of the aforementioned surrogate markers was prognostic. Taken together, P16-IHC as a single biomarker displays the best diagnostic accuracy for prognosis stratification in OPSCC patients with a direct detection of HPV-DNA by PCR or ISH as well as p16-Ki67 dual stain as potential alternatives.

The rough endoplasmic reticulum (ER) of nucleated human cells has crucial functions in protein biogenesis, calcium (Ca2+) homeostasis, and signal transduction. Among the roughly one hundred components, which are involved in protein import and protein folding or assembly, two components stand out: The Sec61 complex and BiP. The Sec61 complex in the ER membrane represents the major entry point for precursor polypeptides into the membrane or lumen of the ER and provides a conduit for Ca2+ ions from the ER lumen to the cytosol. The second component, the Hsp70-type molecular chaperone immunoglobulin heavy chain binding protein, short BiP, plays central roles in protein folding and assembly (hence its name), protein import, cellular Ca2+ homeostasis, and various intracellular signal transduction pathways. For the purpose of this review, we focus on these two components, their relevant allosteric effectors and on the question of how their respective functional cycles are linked in order to reconcile the apparently contradictory features of the ER membrane, selective permeability for precursor polypeptides, and impermeability for Ca2+. The key issues are that the Sec61 complex exists in two conformations: An open and a closed state that are in a dynamic equilibrium with each other, and that BiP contributes to its gating in both directions in cooperation with different co-chaperones. While the open Sec61 complex forms an aqueous polypeptide-conducting- and transiently Ca2+-permeable channel, the closed complex is impermeable even to Ca2+. Therefore, we discuss the human hereditary and tumor diseases that are linked to Sec61 channel gating, termed Sec61-channelopathies, as disturbances of selective polypeptide-impermeability and/or aberrant Ca2+-permeability.

Abstract Background Chromosome 3q26 amplifications have been shown to represent a frequent alteration in various cancer entities including breast cancer. SEC62 - a 3q26 encoded gene - was identified as a potential oncogene and tumor-driver-gene for the pathogenesis of breast cancer. Although the precise physiological function of the respective protein Sec62 is not completely understood, it is hypothesized, that Sec62 induces an increased stress tolerance and enhances cell migration in SEC62 overexpressing cells resulting in a high rate of lymphatic metastasis and poorer overall prognosis in tumor tissue with a high Sec62 expression. We sought to further elucidate the function of Sec62 in breast cancer with special focus on its predictive role on the response to neoadjuvant chemotherapy. Materials and Methods All patients treated for primary breast cancer with neoadjuvant chemotherapy at the Department of Gynaecology and Obstetrics, Saarland University Hospital, Homburg, Germany between 01/2007 and 12/2018 were enrolled in this study. The study was approved by the Saarland institutional review board. Sec62 protein levels were analyzed in tumor tissue samples using immunohistochemistry (IHC). Sec62 immunoreactivity was evaluated by three independent examiners using the immunoreactive score (IRS) according to Remmele and Stegner (0-12). For the assessment of Sec62 protein expression in tumor cells, we rated Sec62 “negative” 0-3, “low” for a score of 4-8 and “high” for 9-12 as described in previous publications. IHC was performed on initial breast core biopsy (CB) tissue (pre-treatment) and on definite pathology specimen (PS) obtained during breast surgery after completion of neoadjuvant chemotherapy. Sec62 expression in both samples were compared and correlated with response to neoadjuvant chemotherapy evaluated pathologically using the semi-quantitative response to neoadjuvant chemotherapy scoring system by Sinn et al. ranging from 0 (no effect) to 4 (no tumor detectable). Additionally, Sec62 expression in tumor tissue was compared with the histologically tumor-free tissue of the same patient. Sec62 expression levels and correlation with response to neoadjuvant chemotherapy were compared using Wilcoxon signed-rank, Mann-Whitney and Pearson`s chi square test. Results 203 patients were assessed for eligibility. Nine cases were excluded, due to incomplete clinical information or insufficient slide quality, leaving 194 patients for final analysis. Median patient age was 54 (22-86) years and median response to neoadjuvant chemotherapy score of 2 (0-4). 20 patients (10 %) had luminal A, 46 (24 %) luminal B, 60 (31 %) Her2/neu positive and 68 (35 %) triple negative breast cancer. All analyzed slides showed an over-expression of Sec62 in breast cancer cells but no positive staining of physiologic breast tissue cells. Median Sec62 expression in core biopsies (CB) (8 (2-12)) was significantly higher compared to expression in final specimen (PS) (4 (0-12); p ≤ 0.01). Median difference between Sec62 expression of CB and PS was 2 (0-4). Regarding response to neoadjuvant chemotherapy patients with a low Sec62 expression in PS and a difference ≥ 6 between CB and PS showed a significant higher median response score compared to other patients (2 (0-4) vs. 1 (0-2); p ≤ 0.01; 4 (1-4) vs. 1 (0-4); p ≤ 0.01). When looking at breast cancer subtypes these effects were strongest in Her2/positive and triple negative patients and median Sec62 expression showed the highest decrease between CB and PS in these two subgroups (6 (-3-12); 2 (-6-12)). Conclusion We identified Sec62 as a potential biomarker for prediction of response to neoadjuvant chemotherapy in patients with primary breast cancer. This effect was observed to be strongest in patients with Her2/positive and triple negative breast cancer. Citation Format: Julia Caroline Radosa, Mariz Kasoha, Merle Doerk, Barbara Linxweiler, Maximilian Linxweiler, Florian Bochen, Rainer M Bohle, Matthias Wagner, Marc P Radosa, Erich-Franz Solomayer, Zoltan Ferenc Takacs. Role of Sec62 in prediction of response to neoadjuvant chemotherapy in patients with primary breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-28.

Background Chromosome 3q26 amplifications have been shown to represent a frequent alteration in various cancer entities including breast cancer. SEC62 - a 3q26 encoded gene - was identified as a potential oncogene and tumor-driver-gene for the pathogenesis of breast cancer. Although the precise physiological function of the respective protein Sec62 is not completely understood, it is hypothesized, that Sec62 induces an increased stress tolerance and enhances cell migration in SEC62 overexpressing cells resulting in a high rate of lymphatic metastasis and poorer overall prognosis in tumor tissue with a high Sec62 expression. We sought to further elucidate the function of Sec62 in breast cancer with special focus on its predictive role on the response to neoadjuvant chemotherapy. Materials and Methods All patients treated for primary breast cancer with neoadjuvant chemotherapy at the Department of Gynaecology and Obstetrics, Saarland University Hospital, Homburg, Germany between 01/2007 and 12/2018 were enrolled in this study. The study was approved by the Saarland institutional review board. Sec62 protein levels were analyzed in tumor tissue samples using immunohistochemistry (IHC). Sec62 immunoreactivity was evaluated by three independent examiners using the immunoreactive score (IRS) according to Remmele and Stegner (0-12). For the assessment of Sec62 protein expression in tumor cells, we rated Sec62 “negative” 0-3, “low” for a score of 4-8 and “high” for 9-12 as described in previous publications. IHC was performed on initial breast core biopsy (CB) tissue (pre-treatment) and on definite pathology specimen (PS) obtained during breast surgery after completion of neoadjuvant chemotherapy. Sec62 expression in both samples were compared and correlated with response to neoadjuvant chemotherapy evaluated pathologically using the semi-quantitative response to neoadjuvant chemotherapy scoring system by Sinn et al. ranging from 0 (no effect) to 4 (no tumor detectable). Additionally, Sec62 expression in tumor tissue was compared with the histologically tumor-free tissue of the same patient. Sec62 expression levels and correlation with response to neoadjuvant chemotherapy were compared using Wilcoxon signed-rank, Mann-Whitney and Pearson`s chi square test. Results 203 patients were assessed for eligibility. Nine cases were excluded, due to incomplete clinical information or insufficient slide quality, leaving 194 patients for final analysis. Median patient age was 54 (22-86) years and median response to neoadjuvant chemotherapy score of 2 (0-4). 20 patients (10 %) had luminal A, 46 (24 %) luminal B, 60 (31 %) Her2/neu positive and 68 (35 %) triple negative breast cancer. All analyzed slides showed an over-expression of Sec62 in breast cancer cells but no positive staining of physiologic breast tissue cells. Median Sec62 expression in core biopsies (CB) (8 (2-12)) was significantly higher compared to expression in final specimen (PS) (4 (0-12); p ≤ 0.01). Median difference between Sec62 expression of CB and PS was 2 (0-4). Regarding response to neoadjuvant chemotherapy patients with a low Sec62 expression in PS and a difference ≥ 6 between CB and PS showed a significant higher median response score compared to other patients (2 (0-4) vs. 1 (0-2); p ≤ 0.01; 4 (1-4) vs. 1 (0-4); p ≤ 0.01). When looking at breast cancer subtypes these effects were strongest in Her2/positive and triple negative patients and median Sec62 expression showed the highest decrease between CB and PS in these two subgroups (6 (-3-12); 2 (-6-12)). Conclusion We identified Sec62 as a potential biomarker for prediction of response to neoadjuvant chemotherapy in patients with primary breast cancer. This effect was observed to be strongest in patients with Her2/positive and triple negative breast cancer. Citation Format: Julia Caroline Radosa, Mariz Kasoha, Merle Doerk, Barbara Linxweiler, Maximilian Linxweiler, Florian Bochen, Rainer M Bohle, Matthias Wagner, Marc P Radosa, Erich-Franz Solomayer, Zoltan Ferenc Takacs. Role of Sec62 in prediction of response to neoadjuvant chemotherapy in patients with primary breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-28.

AbstractObjectiveEndoscopic sinus surgery represents the gold standard for surgical treatment of chronic sinus diseases. Thereby, navigation systems can be of distinct use. In our study, we tested the recently developed KARL STORZ NAV1 SinusTracker navigation software that incorporates elements of augmented reality (AR) to provide a better preoperative planning and guidance during the surgical procedure.MethodsOne hundred patients with chronic sinus disease were operated on using either a conventional navigation software (n = 52, non‐AR, control group) or a navigation software incorporating AR elements (n = 48, AR, intervention group). Incidence of postoperative complications, duration of surgery, surgeon‐reported benefit from the navigation system and patient‐reported postoperative rehabilitation were assessed.ResultsThe surgeons reported a higher benefit during surgery, used the navigation system for more surgical steps and spent longer time with preoperative image analysis when using the AR system as compared with the non‐AR system. No significant differences were seen in terms of postoperative complications, target registration error, operation time and postoperative rehabilitation.ConclusionThe AR enhanced navigation software shows a high acceptance by sinus surgeons in different stages of surgical training and offers potential benefits during surgery without affecting the duration of the operation or the incidence of postoperative complications.Level of evidence1b.