Anti‐EGFR antibody‐drug conjugate for triple‐negative breast cancer therapy

Triple‐negative breast cancers (TNBCs) are highly aggressive, metastatic and recurrent. Cytotoxic chemotherapies with limited clinical benefits and severe side effects are the standard therapeutic strategies, but, to date, there is no efficacious targeted therapy. Literature and our data showed that epidermal growth factor receptor (EGFR) is overexpressed on TNBC cell surface and is a promising oncological target. The objective of this study was to develop an antibody‐drug conjugate (ADC) to target EGFR⁺ TNBC and deliver high‐potency drug. First, we constructed an ADC by conjugating anti‐EGFR monoclonal antibody with mertansine which inhibits microtubule assembly via linker Sulfo‐SMCC. Second, we confirmed the TNBC‐targeting specificity of anti‐EGFR ADC by evaluating its surface binding and internalization in MDA‐MB‐468 cells and targeting to TNBC xenograft in subcutaneous mouse mode. The live‐cell and live‐animal imaging with confocal laser scanning microscopy and In Vivo Imaging System (IVIS) confirmed the TNBC‐targeting. Finally, both in vitro toxicity assay and in vivo anti‐cancer efficacy study in TNBC xenograft models showed that the constructed ADC significantly inhibited TNBC growth, and the pharmacokinetics study indicated its high circulation stability. This study indicated that the anti‐EGFR ADC has a great potential to against TNBC.     

FBXW7 suppresses epithelial‐mesenchymal transition and chemo‐resistance of non‐small‐cell lung cancer cells by targeting snai1 for ubiquitin‐dependent degradation

Objectives

FBXW7 acts as a tumour suppressor by targeting at various oncoproteins for ubiquitin‐mediated degradation. However, the clinical significance and the involving regulatory mechanisms of FBXW7 manipulation of NSCLC regeneration and therapy response are not clear.

Materials and Methods

Immunohistochemical staining and qRT‐PCR were applied to detect FBXW7 and Snai1 expression in 100 samples of NSCLC and matched tumour‐adjacent tissues. FBXW7 manipulation of cancer biological functions were studied by using MTT assay, immunoblotting, flow cytometry, transwells, wound healing assay, and sphere‐formation assays. Immunofluorescence and co‐immunoprecipitation were used to analyse the possible interaction between Snai1 and FBXW7.

Results

We detected the decreased FBXW7 expression in majority of the NSCLC tissues, and lower FBXW7 level was correlated with advanced TNM stage. Furthermore, those patients with decreased FBXW7 expression tend to have both poorer 5‐year survival outcomes, and shorter disease‐free survival, comparing to those with higher FBXW7 levels. Functionally, we found that FBXW7 enforcement suppressed NSCLC progression by inducing cell growth arrest, increasing chemo‐sensitivity and inhibiting Epithelial‐mesenchymal Transition (EMT) progress. Results further showed that FBXW7 could interact with Snai1 directly to degrade its expression through ubiquitylating alternation in NSCLC, which could be partially abrogated by restoring Snai1 expression.

Conclusions

FBXW7 conduction of tumour suppression was partly through degrading Snai1 directly for ubiquitylating regulation in NSCLC

     

A genome‐scale screen reveals context‐dependent ovarian cancer sensitivity to miRNA overexpression

Large‐scale molecular annotation of epithelial ovarian cancer (EOC) indicates remarkable heterogeneity in the etiology of that disease. This diversity presents a significant obstacle against intervention target discovery. However, inactivation of miRNA biogenesis is commonly associated with advanced disease. Thus, restoration of miRNA activity may represent a common vulnerability among diverse EOC oncogenotypes. To test this, we employed genome‐scale, gain‐of‐function, miRNA mimic toxicity screens in a large, diverse spectrum of EOC cell lines. We found that all cell lines responded to at least some miRNA mimics, but that the nature of the miRNA mimics provoking a response was highly selective within the panel. These selective toxicity profiles were leveraged to define modes of action and molecular response indicators for miRNA mimics with tumor‐suppressive characteristics in vivo. A mechanistic principle emerging from this analysis was sensitivity of EOC to miRNA‐mediated release of cell fate specification programs, loss of which may be a prerequisite for development of this disease.     

Peptide ligand‐mediated endocytosis of nanoparticles to cancer cells: Cell receptor‐binding‐ versus cell membrane‐penetrating peptides

The endocytosis‐mediating performances of two types of peptide ligands, cell receptor binding peptide (CRBP) and cell membrane penetrating peptide (CMPP), were analyzed and compared using a common carrier of peptide ligands‐human ferritin heavy chain (hFTH) nanoparticle. Twenty‐four copies of a CMPP(human immunodeficiency virus‐derived TAT peptide) and/or a CRBP (peptide ligand with strong and specific affinity for either human integrin(α_vβ₃) or epidermal growth factor receptor I (EGFR) that is overexpressed on various cancer cells) were genetically presented on the surface of each hFTH nanopariticle. The quantitative level of endocytosis and intracellular localization of fluorescence dye‐labeled CRBP‐ and CMPP‐presenting nanoparticles were estimated in the in vitro cultures of integrin‐ and EGFR‐overexpressing cancer and human dermal fibroblast cells(control). From the cancer cell cultures treated with the CMPP‐ and CRBP‐presenting nanoparticles, it was notable that CRBPs resulted in quantitatively higher level of endocytosis than CMPP (TAT) and successfully transported the nanoparticles to the cytosol of cancer cells depending on concentration and treatment period of time, whereas TAT‐mediated endocytosis localized most of the nanoparticles within endosomal vesicles under the same conditions. These novel findings provide highly useful informations to many researchers both in academia and in industry who are interested in developing anticancer drug delivery systems/carriers.     

Controllable microfluidic synthesis of multiphase drug‐carrying lipospheres for site‐targeted therapy

We report the production of micrometer‐sized gas‐filled lipospheres using digital (droplet‐based) microfluidics technology for chemotherapeutic drug delivery. Advantages of on‐chip synthesis include a monodisperse size distribution (polydispersity index (σ) values of <5%) with consistent stability and uniform drug loading. Photolithography techniques are applied to fabricate novel PDMS‐based microfluidic devices that feature a combined dual hydrodynamic flow‐focusing region and expanding nozzle geometry with a narrow orifice. Spherical vehicles are formed through flow‐focusing by the self‐assembly of phospholipids to a lipid layer around the gas core, followed by a shear‐induced break off at the orifice. The encapsulation of an extra oil layer between the outer lipid shell and inner bubble gaseous core allows the transport of highly hydrophobic and toxic drugs at high concentrations. Doxorubicin (Dox) entrapment is estimated at 15 mg mL^?1 of particles packed in a single ordered layer. In addition, the attachment of targeting ligands to the lipid shell allows for direct vehicle binding to cancer cells. Preliminary acoustic studies of these monodisperse gas lipospheres reveal a highly uniform echo correlation of greater than 95%. The potential exists for localized drug concentration and release with ultrasound energy. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Rho‐associated kinases in tumorigenesis: re‐considering ROCK inhibition for cancer therapy

The Rho‐associated (ROCK) serine/threonine kinases have emerged as central regulators of the actomyosin cytoskeleton, their main purpose being to promote contractile force generation. Aided by the discovery of effective inhibitors such as Y27632, their roles in cancer have been extensively explored with particular attention focused on motility, invasion and metastasis. Recent studies have revealed a surprisingly diverse range of functions of ROCK. These insights could change the way ROCK inhibitors might be used in cancer therapy to include the targeting of stromal rather than tumour cells, the concomitant blocking of ROCK and proteasome activity in K‐Ras‐driven lung cancers and the combination of ROCK with tyrosine kinase inhibitors for treating haematological malignancies such as chronic myeloid leukaemia. Despite initial optimism for therapeutic efficacy of ROCK inhibition for cancer treatment, no compounds have progressed into standard therapy so far. However, by carefully defining the key cancer types and expanding the appreciation of ROCK's role in cancer beyond being a cell‐autonomous promoter of tumour cell invasion and metastasis, the early promise of ROCK inhibitors for cancer therapy might still be realized.     

Clinical relevance and functional implications for human leucocyte antigen‐g expression in non‐small‐cell lung cancer

HLA‐G has been documented both in establishment of anti‐tumour immune responses and in tumour evasion. To investigate the clinical relevance of HLA‐G in non‐small‐cell lung cancer (NSCLC), expression status and potential significance of HLA‐G in NSCLC were analysed. In this study, HLA‐G expression in 101 NSCLC primary lesions and plasma soluble HLA‐G (sHLA‐G) from 91 patients were analysed with immunohistochemistry and ELISA, respectively. Correlations between HLA‐G status and various clinical parameters including survival time were evaluated. Meanwhile, functional analysis of transfected cell surface HLA‐G expression and plasma sHLA‐G form NSCLC patients on natural killer (NK) cell cytolysis were performed. Data revealed that HLA‐G was expressed in 41.6% (42/101) NSCLC primary lesions, while undetectable in adjacent normal lung tissues. HLA‐G expression in NSCLC lesions was strongly correlated to disease stages (P= 0.002). Plasma sHLA‐G from NSCLC patients was markedly higher than that in normal controls (P= 0.004), which was significantly associated with the disease stages (I versus IV, P= 0.025; II versus IV, P= 0.029). Patient plasma sHLA‐G level (≥median, 32.0 U/ml) had a significantly shorter survival time (P= 0.044); however, no similar significance was observed for the lesion HLA‐G expression. In vitro data showed that both cell surface HLA‐G and patient plasma sHLA‐G could dramatically decrease the NK cell cytolysis. Our findings indicated that both lesion HLA‐G expression and plasma sHLA‐G in NSCLC is related to the disease stage and can exert immunosuppression to the NK cell cytolysis, indicating that HLA‐G could be a potential therapeutic target. Moreover, plasma sHLA‐G in NSCLC patients could be used as a prognosis factor for NSCLC.     

Polarization‐dependent second‐harmonic generation for collagen‐based differentiation of breast cancer samples

Nonlinear optical imaging techniques have been widely used to reveal biological structures for accurate diagnosis at the cellular as well as the tissue level. In the present study, polarization‐dependent second‐harmonic generation (PSHG) was used to determine collagen orientation in breast cancer biopsy tissues (grades 0, I, II and III). The obtained data were processed using fast Fourier transform (FFT) analysis, while second‐harmonic generation (SHG) anisotropy and the “ratio parameter” values were also calculated. Such measurements were shown to be able to distinguish collagen structure modifications in different cancer grades tested. The analysis presented herein suggests that PSHG imaging could provide a quantitative evaluation of the tumor state and the distinction of malignant from benign breast tissues. The obtained results also allowed the development of a biophysical model, which can explain the aforementioned differentiations and is in agreement with the simulations relating the SHG anisotropy values with the mechanical tension applied to the collagen during cancer progression. The current approach could be a step forward for the development of new, nondestructive, label free optical diagnostic tools for cancer reducing the need of recalls and unnecessary biopsies, while potentially improving cancer detection rates.     

Genome‐wide identification of urinary cell‐free microRNAs for non‐invasive detection of bladder cancer

Urinary microRNAs (miRNAs) are emerging as clinically useful tool for early and non‐invasive detection of various types of cancer including bladder cancer (BCA). In this study, 205 patients with BCA and 99 healthy controls were prospectively enrolled. Expression profiles of urinary miRNAs were obtained using Affymetrix miRNA microarrays (2578 miRNAs) and candidate miRNAs further validated in independent cohorts using qRT‐PCR. Whole‐genome profiling identified 76 miRNAs with significantly different concentrations in urine of BCA compared to controls (P < 0.01). In the training and independent validation phase of the study, miR‐31‐5p, miR‐93‐5p and miR‐191‐5p were confirmed to have significantly higher levels in urine of patients with BCA in comparison with controls (P < 0.01). We further established 2‐miRNA‐based urinary DxScore (miR‐93‐5p, miR‐31‐5p) enabling sensitive BCA detection with AUC being 0.84 and 0.81 in the training and validation phase, respectively. Moreover, DxScore significantly differed in the various histopathological subgroups of BCA and decreased post‐operatively. In conclusion, we identified and independently validated cell‐free urinary miRNAs as promising biomarkers enabling non‐invasive detection of BCA.     

Identification of Vaccinia‐H1 Related Phosphatase as an Anticancer Target for 1,2,3,4,6‐O‐Pentagalloylglucose

Protein tyrosine phosphatases are involved in diverse human diseases, including cancer, diabetes and inflammatory disorders. Loss of Vaccinia‐H1 related phosphatase (VHR) has been shown to arrest at the G1‐S and G2‐M transitions of the cell cycle, and to increases cell death of prostate cancer cells through JNK activation, suggesting that VHR can be considered as an anticancer target. In this study, 658 natural products were screened through in vitro enzyme assay to identify VHR inhibitor. Among the VHR‐inhibitory compounds, 1,2,3,4,6‐O‐pentagalloylglucose (PGG) was selected for further study as it has been reported to show antitumor effects against tumor model mice, but its direct target has not been identified. PGG inhibited the catalytic activity of VHR (K_i=53 nm ) in vitro. Furthermore, the incubation of HeLa cervical cancer cells with PGG dramatically decreased cell viability and markedly increased the protein levels of the cleaved PARP, a hallmark of apoptosis. In addition, treatment of HeLa cells with PGG significantly reduced the protein levels of cyclin D1, Bcl‐2 and STAT3 phosphorylation. Taken together, these results suggest that PGG could be a potential therapeutic candidate for the treatment of cervical cancer through VHR inhibition.     

Circulating microRNAs,miR‐92a,miR‐100 and miR‐143, as non‐invasive biomarkers for bladder cancer diagnosis

The application of microRNAs (miRNAs) as potential biomarkers and therapy targets has been widely investigated in many kinds of cancers. Recent advantages of serum miRNAs open a new realm of possibilities for non‐invasive diagnosis and prognosis of bladder cancer (BC). The aim of our study was to identify plasma miR‐92a, miR‐100 and miR‐143 expression signatures in patients with BC to introduce new markers for establishing BC diagnosis and prognosis. Blood samples were collected from 70 BC patients and 62 controls. An expression of three target miRNAs (miR‐92a, miR‐100 and miR‐143) was measured using quantitative real‐time PCR method. Results were correlated with clinicopathological data and analysed. Plasma levels of miR‐92a, miR‐100 and miR‐143 were significantly lower in BC patients than in control group. Receiver operator characteristic analysis revealed that the sensitivity and specificity values of miR‐92a were 97·1% and 76·7%, respectively, with a cut‐off value of 0·573. The sensitivity and specificity values of miR‐100 were 90% and 66·7%, respectively, with a cut‐off value of 0·644. The sensitivity and specificity values of miR‐143 were 78·6% and 93·3%, respectively, with a cut‐off value of 0·164. This study explores the existence of specific plasma miRNAs as early diagnostic biomarkers for BC in Egyptian patients; and these findings suggest that plasma miR‐92a, miR‐100 and miR‐143 could be promising novel circulating biomarkers in clinical detection of BC. Copyright © 2016 John Wiley & Sons, Ltd.     

Label‐free diagnosis for colorectal cancer through coffee ring‐assisted surface‐enhanced Raman spectroscopy on blood serum

Surface‐enhanced Raman spectroscopy (SERS) is garnering considerable attention for the swift diagnosis of pathogens and abnormal biological status, that is, cancers. In this work, a simple, fast and inexpensive optical sensing platform is developed by the design of SERS sampling and data analysis. The pretreatment of spectral measurement employed gold nanoparticle colloid mixing with the serum from patients with colorectal cancer (CRC). The droplet of particle‐serum mixture formed coffee‐ring‐like region at the rim, providing strong and stable SERS profiles. The obtained spectra from cancer patients and healthy volunteers were analyzed by unsupervised principal component analysis (PCA) and supervised machine learning model, such as support‐vector machine (SVM), respectively. The results demonstrate that the SVM model provides the superior performance in the classification of CRC diagnosis compared with PCA. In addition, the values of carcinoembryonic antigen from the blood samples were compiled with the corresponding SERS spectra for SVM calculation, yielding improved prediction results.