Statin use and risk of nonmelanoma skin cancer: a nationwide study in Denmark

Background:

Evidence is conflicting regarding statin use and risk of basal cell (BCC) and squamous cell skin cancer (SCC).

Methods:

Using Danish nationwide registries, we identified all patients with incident BCC/SCC during 2005–2009 and matched them to population controls. We computed odds ratios (ORs) for BCC and SCC associated with statin use.

Results:

We identified 38 484 cases of BCC and 3724 cases of SCC. Statin ever use was associated with ORs of 1.09 (CI: 1.06–1.13) for BCC and 1.01 (CI: 0.91–1.11) for SCC.

Conclusions:

Statin use was not associated with risk of SCC. Residual confounding plausibly explains the marginally increased risk of BCC.     

Expression of laminin 5-γ2 chain in cutaneous squamous cell carcinoma and its role in tumour invasion

Background:

Laminin-5 (Ln5), a heterotrimer composed of three chains (α3, β3, and γ2), is a major component of the basement membrane in most adult tissues. One of the chains, Ln5-γ2, is a marker of invasive tumours because it is frequently expressed as a monomer in malignant tumours. Recent studies from our laboratories detected higher levels of Ln5-γ2 expression in basal cell carcinoma (BCC) than in trichoblastoma. Furthermore, Ln5-γ2 overexpression tended to correlate with aggressiveness in BCC.

Methods:

In this study, we compared the expression of Ln5-γ2 in invasive squamous cell carcinoma (SCC, n=62) of the skin to that in preinvasive Bowen''s disease (BD, n=51), followed by analysis of the role of Ln5-γ2 in cancer invasion in vitro.

Results:

Immunohistochemically, the proportion of SCC cases (86%) strongly positive for Ln5-γ2 expression was higher than that of BD (16%). Real-time RT–PCR showed Ln5-γ2 overexpression in SCC cell line, A431, compared with normal keratinocyte cell line, HaCaT. Ln5-γ2 monomer and proteolytically cleaved, biologically active fragments of Ln5-γ2 were identified in SCC tumour extracts. In in vitro raft cultures, which simulate in vivo conditions, Ln5-γ2 siRNA significantly suppressed epidermal growth factor (EGF)-stimulated A431 cell invasion.

Conclusion:

Our results indicate that Ln5-γ2 has a role in cutaneous SCC invasion.     

In-vivo transfection of pcDNA3.1-IGFBP7 inhibits melanoma growth in mice through apoptosis induction and VEGF downexpression

Background

Genome-wide RNA interference screening study revealed that loss of expression of insulin-like growth factor binding protein 7 (IGFBP7) is a critical step in development of a malignant melanoma (MM), and this secreted protein plays a central role in apoptosis of MM. In this study we constructed pcDNA3.1-IGFBP7 to obtain high expression of IGBPF7 and to inhibit the growth of MM in C57BL/6J mice.

Methods

pcDNA3.1-IGFBP7 was transfected into B16-F10 cell, the expression of IGFBP7 was detected by RT-PCR and western blot. The proliferations and apoptosis rates of transfected and control cells were measured by CCK8 and FCM, respectively. The tumorigenicity and tumor growth in both pcDNA3.1-IGFBP7 group and control groups were studied in C57BL/6J mice model. IGFBP7, caspase-3, and VEGF expressions in tumor tissue were measured by immunohistochemistry. Apoptosis of tumors were detected by TUNEL.

Results

We demonstrated this plasmid inhibited proliferation of B16-F10 melanoma cells efficiently in vivo, exploiting the high expression of IGFBP7. More importantly, in-vivo transfection of pcDNA3.1-IGFBP7 inhibited MM growth in C57BL/6J mice. The inhibition of MM growth was proved owing to apoptosis and reduced expression of VEGF induced by pcDNA3.1-IGFBP7.

Conclusions

These results suggest a potential new clinical strategy for MM gene treatment.     

High-risk human papillomavirus in non-melanoma skin lesions from renal allograft recipients and immunocompetent patients

Background:

High-risk human papillomaviruses (HR-HPVs) can be detected in a proportion of non-melanoma skin cancers. Data on prevalence are inconclusive, but are essential to estimate the relevance of HR-HPV, particularly with regard to prophylactic HPV vaccines for skin cancer prevention.

Methods:

High-risk human papillomavirus DNA was investigated in 140 non-melanoma skin lesions from 54 immunocompetent patients and 33 immunosuppressed renal allograft recipients. Expression of p16^INK4a, a marker for HR-HPV oncogene expression in the uterine cervix, and of p53 and pRB was evaluated immunohistochemically.

Results:

The highest prevalence of HR-HPV was found in squamous cell cancer (SCC) (46.2% (6 out of 13) in immunosuppressed and 23.5% (4 out of 17) in immunocompetent patients). High-risk human papillomavirus positivity was accompanied by diffuse p16^INK4a expression in most SCC (P<0.001) and basal cell cancers (P=0.02), while almost all SCC in situ were p16^INK4a positive irrespective of HR-HPV presence (P=0.66). Diffuse p16^INK4a expression was associated with lack of pRB expression (P=0.001). p53 was strongly expressed in 40.0% (56 out of 140) of the lesions irrespective of HR-HPV presence.

Conclusion:

High-risk human papillomavirus can be detected in lesions of keratinised squamous epithelia. The association of HR-HPV with diffuse p16^INK4a expression might indicate HR-HPV oncogene expression in a proportion of lesions. Overexpression of p53 suggests p53 pathway alterations in HR-HPV-positive and -negative lesions.     

Breast cancer patients' clinical outcome measures are associated with Src kinase family member expression

Background:

This study determined mRNA expression levels for Src kinase family (SFK) members in breast tissue specimens and assessed protein expression levels of prominent SFK members in invasive breast cancer to establish associations with clinical outcome. Ki67 was investigated to determine association between SFK members and proliferation.

Methods:

The mRNA expression levels were assessed for eight SFK members by quantitative real-time PCR. Immunohistochemistry was performed for c-Src, Lyn, Lck and Ki67.

Results:

mRNA expression was quantified in all tissue samples. SRC and LYN were the most highly expressed in malignant tissue. LCK was more highly expressed in oestrogen receptor (ER)-negative, compared with ER-positive tumours. High cytoplasmic Src kinase protein expression was significantly associated with decreased disease-specific survival. Lyn was not associated with survival at any cellular location. High membrane Lck expression was significantly associated with improved survival. Ki67 expression correlated with tumour grade and nuclear c-Src, but was not associated with survival.

Conclusions:

All eight SFK members were expressed in different breast tissues. Src kinase was highest expressed in breast cancer and had a negative impact on disease-specific survival. Membrane expression of Lck was associated with improved clinical outcome. High expression of Src kinase correlated with high proliferation.     

Human Papillomaviruses,p16INK4a and Akt expression in basal cell carcinoma

Background

The pathogenic role of beta-HPVs in non melanoma skin cancer (NMSC), is not still completely understood, and literature data indicate that they might be at least cofactors in the development of certain cutaneous squamous cell carcinomas. However, only few reports contain data on basal cell carcinoma (BCC). The HPVs interact with many cellular proteins altering their function or the expression levels, like the p16^INK4a and Akt. Our study aimed to determine the presence of different beta -HPV types and the expression of p16^INK4a and Akt in BCC, the commonest NMSC, in the normal appearing perilesional skin and in forehead swab of 37 immunocompetent patients.

Methods

The expression of p16^INK4a and Akt, by immunohistochemistry, and the HPV DNA, by nested PCR, were investigated in each sample.

Results

No correspondence of HPV types between BCC and swab samples was found, whereas a correspondence between perilesional skin and BCC was ascertained in the 16,7% of the patients. In BCC, 16 different types of beta HPV were found and the most frequent types were HPV107 (15,4%), HPV100 (11,5%) and HPV15 (11,5%) all belonging to the beta HPV species 2. Immunohistochemistry detected significant p16^INK4a expression in almost all tumor samples (94,3%) with the highest percentages (> 30%) of positive cells detected in 8 cases. A statistically significant (p = 0,012) increase of beta HPV presence was detected in p16^INK4a strongly positive samples, in particular of species 2. pAkt expression was detected in all tumor samples with only 2 cases showing rare positive cells, whereas Akt2 expression was found in 14 out of 35 BCC (40%); in particular in HPV positive samples over-expressing p16^INK4a.

Conclusions

Our data show that p16^INK4a and pAkt are over-expressed in BCC and that the high expression of p16^INK4a and of Akt2 isoform is often associated with the presence of beta-HPV species 2 (i.e. HPV 15). The association of these viruses with the up-regulation of p16^INK4a and Akt/PI3K pathway suggests that in a subtype of BCC these viruses may exert a role in the carcinogenesis or in other, still undefined, biological property of these tumors. If this particular type of BCC reflects a different biology it will remain undisclosed until further studies on a larger number of samples will be performed.     

Exon-array profiling unlocks clinically and biologically relevant gene signatures from formalin-fixed paraffin-embedded tumour samples

Background:

Degradation and chemical modification of RNA in formalin-fixed paraffin-embedded (FFPE) samples hamper their use in expression profiling studies. This study aimed to show that useful information can be obtained by Exon-array profiling archival FFPE tumour samples.

Methods:

Nineteen cervical squamous cell carcinoma (SCC) and 9 adenocarcinoma (AC) FFPE samples (10–16-year-old) were profiled using Affymetrix Exon arrays. The gene signature derived was tested on a fresh-frozen non-small cell lung cancer (NSCLC) series. Exploration of biological networks involved gene set enrichment analysis (GSEA). Differential gene expression was confirmed using Quantigene, a multiplex bead-based alternative to qRT–PCR.

Results:

In all, 1062 genes were higher in SCC vs AC, and 155 genes higher in AC. The 1217-gene signature correctly separated 58 NSCLC into SCC and AC. A gene network centered on hepatic nuclear factor and GATA6 was identified in AC, suggesting a role in glandular cell differentiation of the cervix. Quantigene analysis of the top 26 differentially expressed genes correctly partitioned cervix samples as SCC or AC.

Conclusion:

FFPE samples can be profiled using Exon arrays to derive gene expression signatures that are sufficiently robust to be applied to independent data sets, identify novel biology and design assays for independent platform validation.     

Personal history of rosacea and risk of incident cancer among women in the US

Background:

Rosacea is an inflammatory skin disease. We examined the association between personal history of rosacea and risk of incident cancers.

Methods:

A total of 75 088 whites were included from the Nurses'' Health Study II (1991–2011). Information on clinician-diagnosed rosacea and diagnosis year was collected in 2005. All cancers other than basal cell carcinoma (BCC) were confirmed.

Results:

During 1 447 205 person-years, we identified 5194 cases with internal malignancies and 5788 with skin cancers. We did not observe significant associations between personal history of rosacea and internal malignancies, except for thyroid cancer (hazard ratio (HR)=1.59, 95% confidence interval (CI)=1.07–2.36). Among skin cancers, personal history of rosacea was associated with an elevated risk of BCC (HR=1.50, 95% CI=1.35–1.67).

Conclusions:

We suggest possible associations between personal history of rosacea and an increased risk of thyroid cancer and BCC. Further studies are warranted to replicate our findings and to explore the underlying mechanisms.     

Expression of nuclear survivin in normal skin and squamous cell carcinoma: a possible role in tumour invasion

Background:

Survivin is detected in few adult normal cells and it is highly expressed in cancer. Nuclear survivin facilitates cell cycle entry, whereas the mitochondrial pool protects cells from apoptosis. Survivin is overexpressed in keratinocyte stem cells (KSCs) and protects them from apoptosis.

Methods:

As KSCs are at the origin of squamous cell carcinoma (SCC), we evaluated survivin expression in normal and cancerous skin in vivo by immunohistochemistry and western blotting. HaCaT cells overexpressing survivin and wound-healing assay are used. Analysis of variance and Student''s T-tests are used for statistical analysis.

Results:

Survivin is localised in both the cytoplasm and nucleus of normal adult and young keratinocytes. Nuclear survivin is detected in one every 10 of 11 basal keratinocytes. When present in suprabasal cells, nuclear survivin is coexpressed with K10 but not with K15 or p75-neurotrophin receptor (p75NTR), a transit amplifying cell marker. Nuclear, but not cytoplasmic, survivin expression markedly increases in actinic keratosis and in SCC in situ, as compared with normal epidermis, and it is highest in poorly differentiated SCC. In SCC tumours, nuclear survivin-positive cells are mainly K10/p75NTR-negative and K15-positive. In poorly differentiated tumours, survivin mostly localises in the deep infiltrating areas. When overexpressed in keratinocytes, survivin increases cell migration.

Conclusion:

High survivin expression and the subcellular localisation of survivin correlate with keratinocyte differentiation and are associated with undifferentiated and more invasive SCC phenotype.     

Expression and functional role of CRIPTO-1 in cutaneous melanoma

Background:

CRIPTO-1 (CR-1) is involved in the pathogenesis and progression of human carcinoma of different histological origin. In this study we addressed the expression and the functional role of CR-1 in cutaneous melanoma.

Methods:

Expression of CR-1 protein in melanomas and melanoma cell lines was assessed by immunohistochemistry, western blotting and/or flow cytometry. Levels of mRNA were evaluated by real-time PCR. Invasion assays were performed in Matrigel-coated modified Boyden chambers.

Results:

Expression of CR-1 protein and/or mRNA was found in 16 out of 37 primary human cutaneous melanomas and in 12 out of 21 melanoma cell lines. Recombinant CR-1 protein activated in melanoma cells c-Src and, at lesser extent, Smad signalling. In addition, CR-1 significantly increased the invasive ability of melanoma cells that was prevented by treatment with either the ALK4 inhibitor SB-431542 or the c-Src inhibitor saracatinib (AZD0530). Anti-CR-1 siRNAs produced a significant inhibition of the growth and the invasive ability of melanoma cells. Finally, a close correlation was found in melanoma cells between the levels of expression of CR-1 and the effects of saracatinib on cell growth.

Conclusion:

These data indicate that a significant fraction of cutaneous melanoma expresses CR-1 and that this growth factor is involved in the invasion and proliferation of melanoma cells.     

Immunohistochemical expression of the glucose transporters Glut-1 and Glut-3 in human malignant melanomas and benign melanocytic lesions

Background

Reported data indicate that cancer cells have increased rates of glucose metabolism, as determined by 18FDG-PET imaging in patients with malignancies. The results of many studies have demonstrated that the expression of glucose transporters, especially Glut-1, is increased in a variety of malignancies. This study was undertaken to assess the differential expression of Glut-1 and Glut-3 by benign and malignant melanocytic lesions.

Methods

Immunohistochemical staining for Glut-1 and Glut-3 was performed on paraffin-embedded tissue sections prepared from melanocytic nevi (12 cases), Spitz nevi (12 cases) and primary cutaneous malignant melanomas (20 cases).

Results

We observed immunoreactivity for Glut-1 in all melanocytic nevi, 9 of the 12 Spitz nevi and in 9 of the 20 malignant melanomas, whereas Glut-3 was expressed in all the melanocytic lesions, both benign and malignant.

Conclusion

These findings indicate that the glucose transporters Glut-1 and Glut-3 play a role in the glucose metabolism of melanocytic cells. Glut-1 was present in the majority of benign nevi, whereas its expression was downregulated in 55% of malignant melanomas. Our results suggest that glucose transporter Glut-1 expression can significantly discriminate between human malignant melanoma and benign melanocytic nevi, and support the idea that additional mechanisms other than Glut-1 may contribute to glucose uptake in melanomas.     

Tetraspanin-induced death of myeloma cell lines is autophagic and involves increased UPR signalling

Background:

Multiple myeloma (MM) therapy is hindered by the interaction of the heterogeneous malignant plasma cells with their microenvironment and evolving drug resistance. We have previously shown that the membranal tetraspanins, CD81 and CD82, are under-expressed in MM cells and that their reintroduction causes massive non-apoptotic death. In this study, we aimed to characterise the tetraspanin-induced MM death.

Methods:

Multiple myeloma cell lines were transiently transfected with eGFP–CD81N1/CD82N1 fusion proteins and assessed for death mode by flow cytometry (propidium iodide, ZVAD-fmk, 3MA), activation of unfolded protein response (UPR), and autophagy (immunoblot, RT–PCR).

Results:

Cell death induced by CD81N1 and CD82N1 in MM cell lines was autophagic and involved endoplasmic reticulum (ER)-stress manifested by activation of UPR pathways, PERK (protein kinase-like ER kinase) and IRE1 (inositol-requiring 1). We also established the relative X-box binding protein 1 baseline expression levels in a panel of MM cell lines and their general dependence on autophagy for survival. Timeline of UPR cascades and cell fate supported our results.

Interpretation:

This is the first publication implicating tetraspanins in UPR signalling pathways, autophagy, and autophagic death. Integration of our findings with published data highlights the unifying dependence of MM cells on ER–Golgi homoeostasis, and underscores the potential of tetraspanin complexes and ER-stress as leverage for MM therapy.     

Is there an association with phosphorylation and dephosphorylation of Src kinase at tyrosine 530 and breast cancer patient disease-specific survival

Background:

Recent work has demonstrated that c-Src and fully activated Y419Src expression was associated with poor clinical outcome of breast cancer patients. It is unknown whether different activation stages of c-Src equally influence disease-specific survival of breast cancer patients.

Methods:

Immunohistochemistry was performed on 165 resected breast cancers using antibodies to phosphorylated and dephosphorylated Src kinase tyrosine site 530. Expression was assessed using the weighted histoscore method.

Results:

Majority of phosphorylated and dephosphorylated Y530Src expression was observed in the nucleus and cytoplasm. Only 3.6% of phosphorylated Y530Src (pY530Src) expression was detected in the membrane, compared with 53% with dephosphorylated Y530Src. Nuclear expression of pY530Src correlated negatively with oestrogen receptor (ER) status (χ² P<0.001), whereas cytoplasmic phosphorylated and dephosphorylated Y530Src expression correlated negatively with membrane c-Src expression (χ² P=0.008, χ² P<0.001). On univariate and multivariate analysis, no significant association was noticed between phosphorylated or dephosphorylated Y530Src expression and disease-specific survival at any cellular location.

Conclusion:

ER-negative breast cancer patients were more likely to express pY530Src in the nucleus. Breast cancer patients with higher cytoplasmic expression of phosphorylated or dephosphorylated Y530Src were more likely not to express c-Src at the membrane. Phosphorylated and dephosphorylated Y530Src expression is not associated with survival of patients.     

Abrogated expression of DEC1 during oesophageal squamous cell carcinoma progression is age- and family history-related and significantly associated with lymph node metastasis

Background:

Oesophageal squamous cell carcinoma (SCC) causes the highest number of cancer deaths in some regions of Northern China. Previously, we narrowed down a critical region at 9q33-34, identified to be associated with tumour-suppressive function of deleted in oesophageal cancer 1 (DEC1) in oesophageal SCC.

Methods:

We generated DEC1 antibody and constructed tissue microarrays (TMAs) utilising tissue specimens from Henan, a high-risk region for oesophageal SCC, to investigate the importance of DEC1 expression in this cancer.

Results:

Tissue microarray immunohistochemical staining reveals significant loss of DEC1 from hyperplasia, to tumour, and to lymph node metastasis. In addition, the loss of DEC1 in tumour is age-dependent. Interestingly, there is significant abrogation of DEC1 expression in patients with a family history of oesophageal SCC. Deleted in oesophageal cancer 1 localises to both the cytoplasm and nucleus. The vesicular pattern of DEC1 in the cytoplasm appears to localise at the Golgi and Golgi–endoplasmic reticulum intermediate compartment.

Conclusion:

This is the first TMA study to suggest a clinical association of DEC1 in lymph node metastatic oesophageal SCC, early onset oesophageal SCC and familial oesophageal SCC development. Subcellular localisation of DEC1 and its expression in oesophageal SCC tissues provide important insight for further deciphering the molecular mechanism of DEC1 in oesophageal SCC development.     

PDK1 inhibition is a novel therapeutic target in multiple myeloma

Background:

Cancer cells utilise the glycolytic pathway even when adequate oxygen is present, a phenomenon known as the Warburg effect. We examined whether this system is operative in multiple myeloma (MM) cells and whether glycolysis inhibition is a potential therapeutic modality.

Methods:

The MM cells were purified from 59 patients using CD138-immunomagnetic beads. The expression levels of genes associated with glycolysis, c-MYC, GLUT1, LDHA, HIF1A and pyruvate dehydrogenase kinase-1 (PDK1) were determined by real-time PCR. Glucose consumption and lactate production by MM cell lines were analysed. Oxamate, an LDH inhibitor, and dichloroacetate (DCA), a PDK1 inhibitor, were employed. Inhibition of PDK1 expression was achieved using a siRNA.

Results:

High LDHA expression was found to be an indicator of poor prognosis. It was also positively correlated with the expression of PDK1, c-MYC and GLUT1. Greater glucose consumption and lactate production in MM cells was associated with higher LDHA expression. All the glycolysis inhibitors (oxamate, DCA and PDK1 siRNA) induced apoptosis in MM cells. DCA combined with bortezomib showed additive cytotoxic effects.

Conclusion:

The present data suggest that the Warburg effect is operative in MM cells. As PDK1 is not overexpressed in normal tissues, PDK1 inhibition could serve as a novel therapeutic approach.     

Differential expression of ANXA6, HSP27, PRDX2, NCF2, and TPM4 during uterine cervix carcinogenesis: diagnostic and prognostic value

Background:

Cytology-based diagnostics of squamous cervical cancer (SCC) precursor lesions is subjective and can be improved by objective markers.

Methods:

IHC-based analysis of ANXA6, HSP27, peroxiredoxin 2 (PRDX2), NCF2, and tropomyosin 4 (TPM4) during SCC carcinogenesis.

Results:

Expression of ANXA6, HSP27, PRDX2, and NCF2 in the cytoplasm of dysplastic cells increased from cervical intraepithelial neoplasia 2/3 (CIN2/3) to microinvasive cancer. Invasive SCC showed lower expression of TPM4 than CIN and normal epithelium. CIN2/3 with the highest sensitivity and specificity differed from normal epithelium by cytoplasmic expression of HSP27. Patients with cytoplasmic HSP27 expression in SCC deviating from that observed in normal epithelium had worse relapse-free (P=0.019) and overall (P=0.014) survival. Invasive SCC with the highest sensitivity and specificity differed from normal epithelium by expression of PRDX2 and TPM4 in the cytoplasm, from CIN2/3 by the expression of ANXA6 and TPM4 in the cytoplasm, and from microinvasive SCC by the expression of PRDX2 and ANXA6 in the cytoplasm. The number of sporadic ANXA6+ cells between the atypical cells increased from CIN2/3 to invasive SCC.

Conclusion:

Detection of expression changes of the proteins ANXA6, HSP27, PRDX2, NCF2, and TPM4 in SCC precursor lesions may aid current cytological and pathological diagnostics and evaluation of prognosis.     

Hedgehog/Gli promotes epithelial-mesenchymal transition in lung squamous cell carcinomas

Background

Squamous cell carcinomas (SCC) account for approximately 30% of non-small cell lung cancer. Investigation of the mechanism of invasion and metastasis of lung SCC will be of great help for the development of meaningful targeted therapeutics. This study is intended to understand whether the activation of Hedgehog (Hh) pathway is involved in lung SCC, and whether activated Hh signaling regulates metastasis through epithelial-mesenchymal transition (EMT) in lung SCC.

Methods

Two cohorts of patients with lung SCC were studied. Protein expression was examined by immunohistochemistry, Western blot, or immunofluorescence. Protein expression levels in tissue specimens were scored and correlations were analyzed. Vismodegib and a Gli inhibitor were used to inhibit Shh/Gli activity, and recombinant Shh proteins were used to stimulate the Hh pathway in lung SCC cell lines. Cell migration assay was performed in vitro.

Results

Shh/Gli pathway components were aberrantly expressed in lung SCC tissue samples. Gli1 expression was reversely associated with the expression of EMT markers E-Cadherin and β-Catenin in lung SCC specimens. Inhibition of the Shh/Gli pathway suppressed migration and up-regulated E-Cadherin expression in lung SCC cells. Stimulation of the pathway increased migration and down-regulated E-Cadherin expression in lung SCC cells.

Conclusions

Our results suggested that the Shh/Gli pathway may be critical for lung SCC recurrence, metastasis and resistance to chemotherapy. Inhibition of the Shh/Gli pathway activity/function is a potential therapeutic strategy for the treatment of lung SCC patients.