Maximilian Linxweiler

Sec62 bridges the gap from 3q amplification to molecular cell biology in non-small cell lung cancer.

The molecular carcinogenesis of lung cancer has yet to be clearly elucidated. We investigated the possible oncogenic function of SEC62 in lung cancer, which was predicted based on our previous findings that lung and thyroid cancer tissue samples exhibited increased Sec62 protein levels. The SEC62 gene locus is at 3q26.2, and 3q amplification is reportedly the most common genomic alteration in non-small cell lung cancer. We analyzed SEC62 mRNA and protein levels in tissue samples from lung cancer patients by real-time quantitative PCR, Western blot, and IHC and found significantly increased SEC62 mRNA and protein levels in tumors compared with tumor-free tissue samples from the same patients. Correlation analyses revealed significantly higher Sec62 levels in tumors with lymph node metastases compared with nonmetastatic tumors, as well as in poorly compared with moderately differentiated tumors. On the basis of these promising results, we examined the role of Sec62 in cancer cell biology in vitro. Cell migration assays with lung and thyroid cancer cells showed distinct stimulation of migration in SEC62-overexpressing cells and inhibition of migration in Sec62-depleted cells. Moreover, we found that SEC62 silencing sensitized the cells to thapsigargin-induced endoplasmic reticulum stress. Thus, our results indicate that SEC62 represents a potential candidate oncogene in the amplified 3q region in cases of non-small cell lung cancer and harbors various functions in cancer cell biology.

Targeting cell migration and the endoplasmic reticulum stress response with calmodulin antagonists: a clinically tested small molecule phenocopy of SEC62 gene silencing in human tumor cells

BackgroundTumor cells benefit from their ability to avoid apoptosis and invade other tissues. The endoplasmic reticulum (ER) membrane protein Sec62 is a key player in these processes. Sec62 is essential for cell migration and protects tumor cells against thapsigargin-induced ER stress, which are both linked to cytosolic Ca2+. SEC62 silencing leads to elevated cytosolic Ca2+ and increased ER Ca2+ leakage after thapsigargin treatment. Sec62 protein levels are significantly increased in different tumors, including prostate, lung and thyroid cancer.MethodsIn lung cancer, the influence of Sec62 protein levels on patient survival was analyzed using the Kaplan-Meier method and log-rank test. To elucidate the underlying pathophysiological functions of Sec62, Ca2+ imaging techniques, real-time cell analysis and cell migration assays were performed. The effects of treatment with the calmodulin antagonists, trifluoperazine (TFP) and ophiobolin A, on cellular Ca2+ homeostasis, cell growth and cell migration were compared with the effects of siRNA-mediated Sec62 depletion or the expression of a mutated SEC62 variant in vitro. Using Biacore analysis we examined the Ca2+-sensitive interaction of Sec62 with the Sec61 complex.ResultsSec62 overproduction significantly correlated with reduced patient survival. Therefore, Sec62 is not only a predictive marker for this type of tumor, but also an interesting therapeutic target. The present study suggests a regulatory function for Sec62 in the major Ca2+ leakage channel in the ER, Sec61, by a direct and Ca2+-sensitive interaction. A Ca2+-binding motif in Sec62 is essential for its molecular function. Treatment of cells with calmodulin antagonists mimicked Sec62 depletion by inhibiting cell migration and rendering the cells sensitive to thapsigargin treatment.ConclusionsTargeting tumors that overproduce Sec62 with calmodulin antagonists in combination with targeted thapsigargin analogues may offer novel personalized therapeutic options.

Characterization of miR-146a and miR-155 in blood, tissue and cell lines of head and neck squamous cell carcinoma patients and their impact on cell proliferation and migration

PurposeHead and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies worldwide with an unchanged 5-year survival rate during the last decade. To detect reliable prognostic markers and improve patients’ outcome in future, the aim of our study was to detect differences in microRNA (miRNA; miR) expression profile and further on to analyze the functional role of selected miRNAs.MethodsBlood samples from HNSCC patients and sex- and age-matched healthy volunteers were analyzed by microarrays and validated by quantitative real-time PCR. Data were compared with tumor tissue results and all findings were correlated with clinical parameters. Additionally, the proliferation and migration potential of two cell lines transfected with miRNA mimics and inhibitors for miR-146a and miR-155 were examined.ResultsInitial analysis of blood samples showed no significant differences between the miRNA profile of HNSCC patients and healthy controls (pxa0>xa00.05). Interestingly, down-regulation of miR-146a and miR-155 in blood of patients correlated with the occurrence of distant metastasis regarding tumor patients only (pxa0=xa00.023 and pxa0=xa00.028, respectively). Additionally, our investigations in tissue samples revealed a lower expression of miR-155 in tumor cells (pxa0=xa00.003) and a correlation with higher cT-classification for down-regulation of miR-146a (pxa0=xa00.005). Moreover, functional assays demonstrated that inhibition of miR-146a and miR-155 promoted dramatically proliferation and migration potential, whereas transfection of both mimics had an inhibitory effect.ConclusionsCharacterizing the expression of miR-146a and miR-155 and their functional role in tumor biology underlined significantly their proliferation and migration potential suggesting relevance as potential prognostic markers in HNSCC.

Combination of p16 INK4a -Ki67 Immunocytology and HPV Polymerase Chain Reaction for the Noninvasive Analysis of HPV Involvement in Head and Neck Cancer

We recently read an interesting article by Linxweiler et al, who reported the use of p16-Ki67 immunocytochemistry combined with human papillomavirus (HPV) testing on cytological specimens for the assessment of HPV involvement in head and neck squamous cell carcinoma (HNSCC). Their findings regarding brush cytology and HPV testing are in agreement with the results of our previous study. In fact, we recently reported that abnormal brush cytology is strongly associated with a diagnosis of HNSCC and that HPV testing on cytobrushing specimens from patients with HNSCC is feasible and reliable. Linxweiler et al found a very high sensitivity (98%) and specificity (100%) for head and neck brush cytology, although the estimation of sensitivity based on an analytic population that was composed mostly of patients with cancer (50 of 65 patients) has to be regarded with caution. In fact, a recent work highlighted that disease prevalence affects test characteristics, including sensitivity. In addition, the high sensitivity reported by Linxweiler et al may depend on the fact that the samples were partly obtained during a panendoscopy, a procedure that guarantees a more thorough collection of cytological specimens compared with an ambulatory setting. Indeed, in an ambulatory setting, we collected 100 cytobrushing specimens from patients with HNSCC, and detected abnormal cells in only 77 of them (77%). Linxweiler et al also claimed that false-negative results regarding HPV status assessed on the cytological samples may be avoided by screening the samples for the presence of cancer cells prior to HPV testing. We performed the HPV test on 68 paired cytological-histological specimens from patients with HNSCC (Cohen kappa, 0.84) and based on our results, we only partly agree with the authors. In fact, among 9 morphologically negative cytobrushing specimens that corresponded to HPVpositive tumors, 3 specimens tested negative for HPV (thus representing false-negative results), whereas the other 6 were positive for HPV type 16, a finding that is in agreement with the respective tumor tissue. These findings demonstrate that cytological brushings may yield a reliable HPV test result despite the fact that they do not contain neoplastic cells. Although the performance of head and neck cytology can only be evaluated on a population-based sample, we believe that the study by Linxweiler et al represents a strong contribution toward promoting the use of brush cytology in patients with HNSCC for the assessment of HPV involvement in such tumors.

Combination of p16INK4a/Ki67 immunocytology and hpv polymerase chain reaction for the noninvasive analysis of HPV involvement in head and neck cancer: p16-Ki67 Dual Stain in HNSCC

High‐risk human papillomavirus (HPV) infection has been identified as a relevant risk for the development of head and neck squamous cell carcinomas (HNSCCs). As HPV status has also gained a role as a prognostic and predictive biomarker for this entity, there is a growing demand for valid HPV testing in HNSCC patients

Effect of 3q oncogenes SEC62 and SOX2 on lymphatic metastasis and clinical outcome of head and neck squamous cell carcinomas

Chromosome 3q26 amplification represents a frequent alteration in head and neck squamous cell carcinomas (HNSCCs). Overexpression of 3q26 encoded genes SEC62 and SOX2 was detected in various cancers, including HNSCCs, indicating their potential function as oncogenes. In our study, we elucidated the function of SEC62 and SOX2 in HNSCC patients, with a main focus on their effect on lymphatic metastasis and patient survival. We analyzed SEC62 and SOX2 expression in tissue specimens from 65 HNSCC patients and 29 patients with cervical cancer of unknown primary (CUP); a higher SEC62 and lower SOX2 expression was observed in the lymph node metastases from HNSCC patients compared with the respective primary tumor. Lymph node metastases from CUP patients showed higher SEC62 and lower SOX2 expression compared with lymph node metastases from HNSCC patients. When proceeding from the N1 to the N3 stage, SEC62 expression in the lymph node metastases showed an increase and SOX2 expression showed a decrease. Moreover, both genes showed a highly significant relevance as prognostic biomarkers, with the worst prognosis for patients with high SEC62 and low SOX2 expression levels. In functional analyses, knockdown of SEC62 resulted in an inhibition of HNSCC cell migration while, conversely, SEC62 and SOX2 overexpression stimulated cell migration. Taken together, our study showed that the expression of the 3q oncogenes SEC62 and SOX2 affects lymphatic metastasis and cell migration in HNSCC and CUP patients and has a high prognostic relevance in these diseases.

Let’s talk about Secs: Sec61, Sec62 and Sec63 in signal transduction, oncology and personalized medicine

The heterotrimeric Sec61 complex and the dimeric Sec62/Sec63 complex are located in the membrane of the human endoplasmic reticulum (ER) and play a central role in translocation of nascent and newly synthesized precursor polypeptides into the ER. This process involves targeting of the precursors to the membrane and opening of the polypeptide conducting Sec61 channel for translocation. Apart from this central role in the intracellular transport of polypeptides, several studies of the last decade uncovered additional functions of Sec proteins in intracellular signaling: Sec62 can induce ER-phagy in the process of recovery of cells from ER stress and the Sec61 channel can also act as a passive ER calcium leak channel. Furthermore, mutations, amplifications and an overexpression of the SEC genes were linked to various diseases including kidney and liver diseases, diabetes and human cancer. Studies of the last decade could not only elucidate the functional role of Sec proteins in the pathogenesis of these diseases, but also demonstrate a relevance of Sec62 as a prognostic and predictive biomarker in head and neck cancer, prostate and lung cancer including a basis for new therapeutic strategies. In this article, we review the current understanding of protein transport across the ER membrane as central function of Sec proteins and further focus on recent studies that gave first insights into the functional role and therapeutic relevance of Sec61, Sec62 and Sec63 in human diseases.

Cancer of unknown primary (CUP) of the head and neck: retrospective analysis of 81 patients

The treatment of patients with cervical lymph node metastases without detectable primary tumor remains an important challenge, until today, no standard therapy is available. The present study investigated the multimodal treatment of patients with head and neck CUP syndrome (HNCUP) and their follow-up retrospectively. 81 patients with cervical lymph node metastases without a primary tumor were treated at the Departments of Otorhinolaryngology as well as Radiotherapy and Radiation Oncology at the University of Saarland in Homburg, Germany in the period between 1991 and 2013. All patients received routine work-up consisting of CUP panendoscopy and imaging. Neck dissection was then performed in 77% of the patients. The most common histology was squamous cell carcinoma (80%). Ten percent of the patients had distant metastases. All patients underwent primary or adjuvant radiation therapy, or simultaneous radiochemotherapy. After a median follow-up of 3.5 years, the 5-year survival rate was 30%. There was a local recurrence that was known in 20/63 patients (31%) and distant metastases were documented in 19/61 M0 patients (31%). Higher grade late toxicity (grade 3–4) was observed in 12% of patients. Neck dissection and radiation therapy remains an integral part of HNCUP therapy, while the use of chemotherapy could be considered in selected cases. Prospective multicenter randomized trials would be necessary to identify the best target volume and to clarify the role of chemotherapy.

Identification of SEC62 as a potential marker for 3q amplification and cellular migration in dysplastic cervical lesions

BackgroundChromosome 3 amplification affecting the 3q26 region is a common genomic alteration in cervical cancer, typically marking the transition of precancerous intraepithelial lesions to an invasive phenotype. Though potential 3q encoded target genes of this amplification have been identified, a functional correlation of potential oncogenic function is still missing. In this study, we investigated copy number changes and the expression level of SEC62 encoded at 3q26.2 as a new potential 3q oncogene in dysplastic cervical lesions and analyzed its role in cervical cancer cell biology.MethodsExpression levels of Sec62 and vimentin were analyzed in liquid based cytology specimens from 107 women with varying grades of cervical dysplasia ranging from normal cases to cancer by immunofluorescence cytology. Additionally, a subset of 20 representative cases was used for FISH analyses targeting SEC62. To further explore the functional role of Sec62 in cervical cancer, HeLa cells were transfected with a SEC62 plasmid or SEC62 siRNA and analyzed for their proliferation and migration potential using real-time monitoring and trans-well systems as well as changes in the expression of EMT markers.ResultsFISH analyses of the swabbed cells showed a rising number of SEC62 gains and amplifications correlating to the grade of dysplasia with the highest incidence in high grade squamous intraepithelial lesions and squamous cell carcinomas. When analyzing the expression level of Sec62 and vimentin, we found a gradually increasing expression level of both proteins according to the severity of the dysplasia. In functional analyses, SEC62 silencing inhibited and SEC62 overexpression stimulated the migration of HeLa cells with only marginal effects on cell proliferation, the expression level of EMT markers and the cytoskeleton structure.ConclusionsOur study suggests SEC62 as a target gene of 3q26 amplification and a stimulator of cellular migration in dysplastic cervical lesions. Hence, SEC62 could serve as a potential marker for 3q amplification, providing useful information about the dignity and biology of dysplastic cervical lesions.

Treatment of SEC62 over-expressing tumors by Thapsigargin and Trifluoperazine

Abstract Treatment with analogues of the SERCA-inhibitor Thapsigargin is a promising new approach for a wide variety of cancer entities. However, our previous studies on various tumor cells suggested resistance of SEC62 over-expressing tumors to this treatment. Therefore, we proposed the novel concept that e.g. lung-, prostate-, and thyroid-cancer patients should be tested for SEC62 over-expression, and developed a novel therapeutic strategy for a combinatorial treatment of SEC62 over-expressing tumors. The latter was based on the observations that treatment of SEC62 over-expressing tumor cells with SEC62-targeting siRNAs showed less resistance to Thapsigargin as well as a reduction in migratory potential and that the siRNA effects can be mimicked by the Calmodulin antagonist Trifluoperazine. Therefore, the combinatorial treatment of SEC62 over-expressing tumors was proposed to involve Thapsigargin and Trifluoperazine. Here, we addressed the impact of Thapsigargin and Trifluoperazine in separate and combined treatments of heterotopic tumors, induced by inoculation of human hypopharyngeal squamous cell carcinoma (FaDu)-cells into the mouse flank. Seeding of the tumor cells and/or their growth rate were significantly reduced by all three treatments, suggesting Trifluoperazine is a small molecule to be considered for future therapeutic strategies for patients, suffering from Sec62-overproducing tumors.