High-throughput sequencing of small RNA transcriptomes reveals critical biological features targeted by microRNAs in cell models used for squamous cell cancer research

Abstract Background The implication of post-transcriptional regulation by microRNAs in molecular mechanisms underlying cancer disease is well documented. However, their interference at the cellular level is not fully explored. Functional in vitro studies are fundamental for the comprehension of their role; nevertheless results are highly dependable on the adopted cellular model. Next generation small RNA transcriptomic sequencing data of a tumor cell line and keratinocytes derived from primary culture was generated in order to characterize the microRNA content of these systems, thus helping in their understanding. Both constitute cell models for functional studies of microRNAs in head and neck squamous cell carcinoma (HNSCC), a smoking-related cancer. Known microRNAs were quantified and analyzed in the context of gene regulation. New microRNAs were investigated using similarity and structural search, ab initio classification, and prediction of the location of mature microRNAs within would-be precursor sequences. Results were compared with small RNA transcriptomic sequences from HNSCC samples in order to access the applicability of these cell models for cancer phenotype comprehension and for novel molecule discovery. Results Ten miRNAs represented over 70% of the mature molecules present in each of the cell types. The most expressed molecules were miR-21, miR-24 and miR-205, Accordingly; miR-21 and miR-205 have been previously shown to play a role in epithelial cell biology. Although miR-21 has been implicated in cancer development, and evaluated as a biomarker in HNSCC progression, no significant expression differences were seen between cell types. We demonstrate that differentially expressed mature miRNAs target cell differentiation and apoptosis related biological processes, indicating that they might represent, with acceptable accuracy, the genetic context from which they derive. Most miRNAs identified in the cancer cell line and in keratinocytes were present in tumor samples and cancer-free samples, respectively, with miR-21, miR-24 and miR-205 still among the most prevalent molecules at all instances. Thirteen miRNA-like structures, containing reads identified by the deep sequencing, were predicted from putative miRNA precursor sequences. Strong evidences suggest that one of them could be a new miRNA. This molecule was mostly expressed in the tumor cell line and HNSCC samples indicating a possible biological function in cancer. Conclusions Critical biological features of cells must be fully understood before they can be chosen as models for functional studies. Expression levels of miRNAs relate to cell type and tissue context. This study provides insights on miRNA content of two cell models used for cancer research. Pathways commonly deregulated in HNSCC might be targeted by most expressed and also by differentially expressed miRNAs. Results indicate that the use of cell models for cancer research demands careful assessment of underlying molecular characteristics for proper data interpretation. Additionally, one new miRNA-like molecule with a potential role in cancer was identified in the cell lines and clinical samples.

Carcinoma de células escamosas em glândula mamária de gata doméstica

Background: In the feline species, 80% to 93% of neoplasias in the mammary gland are malignant, being the majority carcinomas. Among them, there is the mammary squamous cell carcinoma, which amounts to a very rare neoplasm in the domestic cat, with considerable potential for malignancy. This study aimed to report a case of squamous cell mammary carcinoma in the feline species. Case: A female cat, mixed breed, ten years old, presented history of skin lesion. The cat had been spayed two years before, but with previous administration of contraceptives. At the physical examination, it was observed ulcer between the caudal abdominal mammary glands. The occurrence of skin or mammary neoplasia was conceived. The following complementary tests were requested: complete blood count, serum biochemical profi le (renal and hepatic), chest radiographs, abdominal ultrasound, and incisional biopsy of the ulcerated region periphery, followed by classic histopathology. The lesion histopathology was compatible with squamous cell carcinoma of the mammary gland. Due to such a diagnosis, bilateral mastectomy was recommended. The material obtained during the surgical procedure was sent for anatomopathological analysis. Microscopically, surgical margins infi ltration and a regional lymph node were verifi ed. The owner was advised of the need for complementary therapies and medical monitoring of the cat. However, there was no return. It is noteworthy that the animal’s physical and laboratory examinations showed no neoplasia in other regions, being the squamous cell carcinoma of the mammary gland considered primary. Discussion: The malignant mammary neoplasia genesis in feline species, in general, seems to be related to steroid hormones. The ovariectomized females are less likely to develop the disease when compared to intact cats, but there is no protective effect of surgery on those spayed after two years of age regarding the appearance of the neoplasia. Thus, at the time the reported patient was ovariectomized, this effect no longer occurred. The synthetic progestins regularly used to prevent estrus increase by three times the risk of breast carcinomas onset. In humans, there is no clear defi nition of the etiology and pathogenesis of mammary squamous cell carcinoma. However, it has been suggested its association with extreme forms of squamous metaplasia present in pre-existing mammary adenocarcinoma, besides cysts, chronic infl ammations, abscesses and mammary gland adenofi bromas. In a hypothetical way, this etiology could also be related to the feline mammary carcinoma, although, for the case at issue, the exogenous and endogenous hormonal infl uence should not be excluded. It has been reported that mammary squamous cell carcinomas in cats are classifi ed in grades II and III (ie, moderately and poorly differentiated, respectively). Thus, they are considered tumors with more unfavorable prognosis. However, the monitoring of the clinical course, in order to evaluate possible recurrence of the neoplasia and metastases to distant sites, was not possible as the animal under discussion did not return. The squamous cell carcinoma is the most common skin tumor in feline species, despite the primary location in the mammary gland. It is, therefore, important to differentiate squamous cell carcinoma originated in the breast from histological types derived from skin. The description of this special and rare feline mammary carcinoma is important due to its particular characteristics and potential for malignancy.

Profili di espressione genetica ed epigenetica ad impatto prognostico e predittivo nel carcinoma squamoso e nelle lesioni potenzialmente maligne del cavo orale

Il carcinoma squamoso orale (CSO) è spesso preceduto da lesioni definite potenzialmente maligne tra cui la leucoplachia e il lichen ma una diagnosi precoce avviene ancora oggi in meno della metà dei casi. Inoltre spesso un paziente trattato per CSO svilupperà secondi tumori. Scopo del lavoro di ricerca è stato: 1) Studiare, mediante metodica di next generation sequencing, lo stato di metilazione di un gruppo di geni a partire da prelievi brushing del cavo orale al fine di identificare CSO o lesioni ad alto rischio di trasformazione maligna. 2) Valurare la relazione esistente tra sovraespressione di p16INK4A e presenza di HPV in 35 pazienti affetti da lichen 3) Valutare la presenza di marker istopatologici predittivi di comparsa di seconde manifestazioni tumorali 4) valutare la relazione clonale tra tumore primitivo e metastasi linfonodale in 8 pazienti mediante 2 metodiche di clonalità differenti: l’analisi di mtDNA e delle mutazioni del gene TP53. I risultati hanno mostrato: 1) i geni ZAP70 e GP1BB hanno presentato un alterato stato di metilazione rispettivamente nel 100% e nel 90,9% di CSO e lesioni ad alto rischio, mentre non sono risultati metilati nei controlli sani; ipotizzando un ruolo come potenziali marcatori per la diagnosi precoce nel CSO. 2)Una sovraespressione di p16INK4A è risultata in 26/35 pazienti affetti da lichen ma HPV-DNA è stato identificato in soli 4 campioni. Nessuna relazione sembra essere tra sovraespressione di p16INK4A e virus HPV. 3)L’invasione perineurale è risultato un marker predittivo della comparsa di recidiva locale e metastasi linfonodale, mentre lo stato dei margini chirurgici si è rilevato un fattore predittivo per la comparsa di secondi tumori primitivi 4) Un totale accordo nei risultati c’è stato tra analisi di mtDNA e analisi di TP53 e le due metodiche hanno identificato la presenza di 4 metastasi linfonodali non clonalmente correlate al tumore primitivo.

Clinicopathologic features and long-term outcomes of NUT midline carcinoma

NUT midline carcinoma (NMC) is a poorly differentiated squamous cancer characterized by rearrangement of the NUT gene. Research advances have provided opportunities for targeted therapy in NMC, yet the clinical features of this rare disease have not been systematically characterized. We report on a large population of such patients to identify the disease characteristics and treatments, correlate them with outcome, and to consider clinical recommendations.

Multicenter phase II trial of preoperative induction chemotherapy followed by chemoradiation with docetaxel and cisplatin for locally advanced esophageal carcinoma (SAKK 75/02)

BACKGROUND: This multicenter phase II study investigated the efficacy and feasibility of preoperative induction chemotherapy followed by chemoradiation and surgery in patients with esophageal carcinoma. PATIENTS AND METHODS: Patients with locally advanced resectable squamous cell carcinoma or adenocarcinoma of the esophagus received induction chemotherapy with cisplatin 75 mg/m(2) and docetaxel (Taxotere) 75 mg/m(2) on days 1 and 22, followed by radiotherapy of 45 Gy (25 x 1.8 Gy) and concurrent chemotherapy comprising cisplatin 25 mg/m(2) and docetaxel 20 mg/m(2) weekly for 5 weeks, followed by surgery. RESULTS: Sixty-six patients were enrolled at eleven centers and 57 underwent surgery. R0 resection was achieved in 52 patients. Fifteen patients showed complete, 16 patients nearly complete and 26 patients poor pathological remission. Median overall survival was 36.5 months and median event-free survival was 22.8 months. Squamous cell carcinoma and good pathologically documented response were associated with longer survival. Eighty-two percent of all included patients completed neoadjuvant therapy and survived for 30 days after surgery. Dysphagia and mucositis grade 3/4 were infrequent (<9%) during chemoradiation. Five patients (9%) died due to surgical complications. CONCLUSIONS: This neoadjuvant, taxane-containing regimen was efficacious and feasible in patients with locally advanced esophageal cancer in a multicenter, community-based setting and represents a suitable backbone for further investigation.

EcPV2 DNA in equine papillomas and in situ and invasive squamous cell carcinomas supports papillomavirus etiology

Equine penile papillomas, in situ carcinomas, and invasive carcinomas are hypothesized to belong to a continuum of papillomavirus-induced diseases. The former ones clinically present as small grey papules, while the latter 2 lesions are more hyperplasic or alternatively ulcerated. To test the hypothesis that these lesions are papillomavirus-induced, samples of 24 horses with characteristic clinical and histologic findings of penile papillomas or in situ or invasive squamous cell carcinomas were collected. As controls, 11 horses with various lesions--namely, Balanoposthitis (6 cases), melanoma (3 cases), follicular cyst (1 case), and amyloidosis (1 case)--were included. DNA was extracted and polymerase chain reaction applied to amplify papillomavirus DNA. The respective primers were designed to amplify DNA of the recently discovered equine papillomavirus EcPV2. All tested papilloma and squamous cell carcinoma samples were found to contain DNA of either of 2 previously published EcPV2 variants. Among the other samples 6 of 11 were found to contain EcPV2 DNA. To further support the findings and to determine where the papillomavirus DNA was located within the lesions, an in situ hybridization for the detection of EcPV2 DNA was established. The samples tested by this technique were found to clearly contain papillomavirus nucleic acid concentrated in the nucleus of the koilocytes. The findings of this study support previous data and the hypothesis that papillomaviruses induce the described penile lesions in horses.

ISL1 expression is not restricted to pancreatic well-differentiated neuroendocrine neoplasms, but is also commonly found in well and poorly differentiated neuroendocrine neoplasms of extrapancreatic origin

The human insulin gene enhancer-binding protein islet-1 (ISL1) is a transcription factor involved in the differentiation of the neuroendocrine pancreatic cells. Recent studies identified ISL1 as a marker for pancreatic well-differentiated neuroendocrine neoplasms. However, little is known about ISL1 expression in pancreatic poorly differentiated and in extrapancreatic well and poorly differentiated neuroendocrine neoplasms. We studied the immunohistochemical expression of ISL1 in 124 neuroendocrine neoplasms. Among pancreatic neuroendocrine neoplasms, 12/13 with poor differentiation were negative, whereas 5/7 with good differentiation but a Ki67 >20% were positive. In extrapancreatic neuroendocrine neoplasms, strong positivity was found in Merkel cell carcinomas (25/25), pulmonary small cell neuroendocrine carcinomas (21/23), medullary thyroid carcinomas (9/9), paragangliomas/pheochromocytomas (6/6), adrenal neuroblastomas (8/8) and head and neck neuroendocrine carcinomas (4/5), whereas no or only weak staining was recorded in pulmonary carcinoids (3/15), olfactory neuroblastomas (1/4) and basaloid head and neck squamous cell carcinomas (0/15). ISL1 stained the neuroendocrine carcinoma component of 5/8 composite carcinomas and also normal neuroendocrine cells in the thyroid, adrenal medulla, stomach and colorectum. Poorly differentiated neuroendocrine neoplasms, regardless of their ISL1 expression, were usually TP53 positive. Our results show the almost ubiquitous expression of ISL1 in extrapancreatic poorly differentiated neuroendocrine neoplasms and neuroblastic malignancies and its common loss in pancreatic poorly differentiated neuroendocrine neoplasms. These findings modify the role of ISL1 as a marker for pancreatic neuroendocrine neoplasms and suggest that ISL1 has a broader involvement in differentiation and growth of neuroendocrine neoplasms than has so far been assumed.

Depressed type 1 cytokine synthesis by superantigen-activated CD4+ T cells of women with human papillomavirus-related high-grade squamous intraepithelial lesions.

Carcinoma of the cervix is causally related to infection with the human papillomavirus (HPV), and T cells play a pivotal role in the immune response of the host to rid itself of HPV infection. Therefore, we assessed the T-cell function of women with HPV-related cervical neoplasia against a superantigen, Staphylococcus enterotoxin B (SEB). Each woman provided a cervical brush specimen for HPV DNA testing and Papanicolaou (Pap) smears for the staging of cervical lesions. They also provided a blood specimen for determination of the ability of CD4(+) T and CD8(+) T cells to synthesize Th1 (interleukin-2 [IL-2], gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and Th2 (IL-10) cytokines in response to activation with SEB. Compared with control subjects with self-attested negative Pap smears, women with high-grade squamous intraepithelial lesions (HSIL) had significantly lower percentages of activated CD4(+) T cells that produced IL-2 (P = 0.045), IFN-gamma (P = 0.040), and TNF-alpha (P = 0.015) and a significantly lower percentage of activated CD8(+) T cells that produced IL-2 (P < 0.01). These data indicate that women with HPV-related cervical HSIL show a decrease in Th1 cytokine production by activated CD4(+) T cells and suggested that compromised T-helper functions may negatively impact the function of cytotoxic CD8(+) T cells.

Prognostic factors and outcomes in primary urethral cancer: results from the international collaboration on primary urethral carcinoma.

PURPOSE To evaluate risk factors for survival in a large international cohort of patients with primary urethral cancer (PUC). METHODS A series of 154 patients (109 men, 45 women) were diagnosed with PUC in ten referral centers between 1993 and 2012. Kaplan-Meier analysis with log-rank test was used to investigate various potential prognostic factors for recurrence-free (RFS) and overall survival (OS). Multivariate models were constructed to evaluate independent risk factors for recurrence and death. RESULTS Median age at definitive treatment was 66 years (IQR 58-76). Histology was urothelial carcinoma in 72 (47 %), squamous cell carcinoma in 46 (30 %), adenocarcinoma in 17 (11 %), and mixed and other histology in 11 (7 %) and nine (6 %), respectively. A high degree of concordance between clinical and pathologic nodal staging (cN+/cN0 vs. pN+/pN0; p < 0.001) was noted. For clinical nodal staging, the corresponding sensitivity, specificity, and overall accuracy for predicting pathologic nodal stage were 92.8, 92.3, and 92.4 %, respectively. In multivariable Cox-regression analysis for patients staged cM0 at initial diagnosis, RFS was significantly associated with clinical nodal stage (p < 0.001), tumor location (p < 0.001), and age (p = 0.001), whereas clinical nodal stage was the only independent predictor for OS (p = 0.026). CONCLUSIONS These data suggest that clinical nodal stage is a critical parameter for outcomes in PUC.

Molecular profiling of lung adenosquamous carcinoma: hybrid or genuine type?

Lung adenosquamous carcinoma is a particular subtype of non-small cell lung carcinoma that is defined by the coexistence of adenocarcinoma and squamous cell carcinoma components. The aim of this study was to assess the mutational profile in each component of 16 adenosquamous carcinoma samples from a Caucasian population by a combination of next generation sequencing using the cancer hotspot panel as well as the colon and lung cancer panel and FISH. Identified mutations were confirmed by Sanger sequencing of DNA from cancer cells of each component collected by Laser Capture microdissection. Mutations typical for adenocarcinoma as well as squamous cell carcinoma were identified. Driver mutations were predominantly in the trunk suggesting a monoclonal origin of adenosquamous carcinoma. Most remarkably, EGFR mutations and mutations in the PI3K signaling pathway, which accounted for 30% and 25% of tumors respectively, were more prevalent while KRAS mutations were less prevalent than expected for a Caucasian population. Surprisingly, expression of classifier miR-205 was intermediate between that of classical adenocarcinoma and squamous cell carcinoma suggesting that adenosquamous carcinoma is a transitional stage between these tumor types. The high prevalence of therapy-relevant targets opens new options of therapeutic intervention for adenosquamous carcinoma patients.

Retinoic receptor gamma in squamous epithelial tumorigenesis

Retinoids, important modulators of squamous epithelial differentiation and proliferation, are effective in the treatment and prevention of squamous epithelial cancers, including squamous cell carcinomas (SCCs) of the skin. However, the mechanism is not well understood. Retinoids exert their effects primarily through two nuclear receptor families, retinoic acid receptors (RARα, β and γ) and retinoid X receptors (RXR(α, β and γ), ligand-dependent DNA-binding transcription factors that are members of the steroid hormone receptor superfamily. Retinoid receptor loss has been correlated with squamous epithelial malignancy. This has lead to the hypothesis that reduced RARγ expression and the resulting suppression of retinoid signaling contributes to squamous epithelial malignancy. To test this hypothesis, I attempted to reduce or abolish expression of RARγ, the predominant RAR in squamous epithelia, in several nontumorigenic human squamous epithelial cell lines. The most useful of these cell lines has been SqCCY1, the human head and neck squamous cell carcinoma cell line, along with several subclones stably transfected with RARγ sense and antisense expression constructs. By several criteria, we observed an overall suppression of squamous differentiation in RARγ sense transfectants and an enhancement in RARγ antisense transfectants, relative to parental SqCCY1 cells. We also observed that both sense and antisense cells could form tumors in athymic mice in vivo, while parental SqCCY1 cells could not. Although these results appear contradictory, several conclusions can be drawn. First, loss of RARγ contributes to squamous epithelial tumorigenesis. Second, overexpression of RARγ leads to tumor formation, suppressing differentiation and promoting proliferation, possibly due to a competitive inhibition of limiting concentrations of RXRα, a common heterodimeric partner for many nuclear receptors in addition to RARs, representing a mechanism for RARγ to modulate squamous epithelial homeostasis. The cause for tumorigenesis in the two conditions is likely due to different mechanisms/roles of RARγ in the cell, with the former as a retinoid signaling regulator; and the latter as an RXRα concentration modulator. Finally, High level of RARγ expression sensitizes cells to environmental RA, enhancing RARγ/RXRα-mediated RA signaling. Therefore, RA should be used in skin lesions with suppressed RARγ expression levels, not in skin lesions with overexpressed RARγ levels. ^

Molecular markers of human papillomavirus (HPV) infection and epidemiologic risk factors for squamous intraepithelial lesions (SIL) of the cervix

Infection with certain types of HPV is a necessary event in the development of cervical carcinoma; however, not all women who become infected will progress. While much is known about the molecular influence of HPV E6 and E7 proteins on the malignant transformation, little is known about the additional factors needed to drive the process. Currently, conventional cervical screening is insufficient at identifying women who are likely to progress from premalignant lesions to carcinoma. Aneuploidy and chromatin texture from image cytometry have been suggested as quantitative measures of nuclear damage in premalignant lesions and cancer, and traditional epidemiologic studies have identified potential factors to aid in the discrimination of those lesions likely to progress. ^ In the current study, real-time PCR was used to quantitate mRNA expression of the E7 gene in women exhibiting normal epithelium, LSIL, and HSIL. Quantitative cytometry was used to gather information about the DNA index and chromatin features of cells from the same women. Logistic regression modeling was used to establish predictor variables for histologic grade based on the traditional epidemiologic risk factors and molecular markers. ^ Prevalence of mRNA transcripts was lower among women with normal histology (27%) than for women with LSIL (40%) and HSIL (37%) with mean levels ranging from 2.0 to 4.2. The transcriptional activity of HPV 18 was higher than that of HPV 16 and increased with increasing level of dysplasia, reinforcing the more aggressive nature of HPV 18. DNA index and mRNA level increased with increasing histological grade. Chromatin score was not correlated with histology but was higher for HPV 18 samples and those with both HPV 18 and HPV 16. However, chromatin score and DNA index were not correlated with mRNA levels. The most predictive variables in the regression modeling were mRNA level, DNA index, parity, and age, and the ROC curves for LSIL and HSIL indicated excellent discrimination. ^ Real-time PCR of viral transcripts could provide a more efficient method to analyze the oncogenic potential within cells from cervical swabs. Epidemiological modeling of malignant progression in the cervix should include molecular markers, as well as the traditional epidemiological risk factors. ^

Examination of synthetic retinoid -induced apoptosis in cutaneous squamous cell carcinomas: Mechanisms and implications for chemoprevention and chemotherapy with retinoids

Non-melanoma skin cancers, including basal cell carcinoma and squamous cell carcinoma (SCC), are the most common neoplasms in the United States with a lifetime risk nearly equal to all other types of cancer combined. Retinoids are naturally occurring and synthetic analogues of vitamin A that bind to nuclear retinoid receptors and modulate gene expression as a means of regulating cell proliferation and differentiation. Retinoids have been employed for many years in the treatment of various cutaneous lesions and for cancer chemoprevention and therapy. The primary drawback limiting the use of retinoids is their toxicity, which is also associated with receptor-gene interactions. In this study, the effects of the synthetic retinoids N-(4-hydroxyphenyl)retinamide (4HPR) and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) were examined in cutaneous keratinocytes. Four human cutaneous SCC cell lines were examined along with normal human epidermal keratinocyte (NHEK) cells from two donors. Sensitivity to 4HPR or CD437 alone or in combination with other agents was determined via growth inhibition, cell cycle distributions, or apoptosis induction. Both synthetic retinoids were able to promote apoptosis in SCC cells more effectively than the natural retinoid all-trans retinoic acid. Apoptosis could not be inhibited by nuclear retinoic acid receptor antagonists. In NHEK cells, 4HPR induced apoptosis while CD437 promoted G1 arrest. 4HPR acted as a prooxidant by generating reactive oxygen species (ROS) in SCC and NHEK cells. 4HPR-induced apoptosis in SCC cells could be inhibited or potentiated by manipulating cellular defenses against oxidative stress, indicating an essential role for ROS in 4HPR-induced apoptosis. CD437 promoted apoptosis in SCC cells in S and G2/M phases of the cell cycle within two hours of treatment, and this rapid induction could not be blocked with cycloheximide. This study shows: (1) 4HPR- and CD437-induced apoptosis do not directly involve a traditional retinoid pathway; (2) 4HPR can act as a prooxidant as a means of promoting apoptosis; (3) CD437 induces apoptosis in SCC cells independent of protein synthesis and is potentially less toxic to NHEK cells; and (4) 4HPR and CD437 operate under different mechanisms with respect to apoptosis induction and this may potentially enhance their therapeutic index in vivo. ^

Transformación de liquen plano oral en carcinoma espinocelular : caso clínico

Fil: Salomón, Susana Elsa. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas

Virus del papiloma humano : presentación oral y su relación con el carcinoma espinocelular

El virus del papiloma humano (VPH) (HPV en su sigla en inglés) es un virus ADN con especial afinidad por células epiteliales, tanto cutáneas como mucosas, que incluyen el epitelio del cérvix, región anogenital y orofaríngea. Existen más de 120 subtipos de VPH subdivididos en dos grupos según su bajo o alto riesgo de inducir actividad oncogénica. En cavidad oral la manifestación clínica benigna de la infección es el papiloma plano.Sin embargo, un 12% de los carcinoma~ o rofaríngeos son causados por este virus. Se ha demostrado que la infección por VPH tiene un papel independiente como factor de riesgo para el desarrollo de carcinoma espinocelular en cavidad oral.