Targeting cancer stemness mediated by BMI1 and MCL1 for non-small cell lung cancer treatment.

Lung cancer is the leading cause of cancer-associated death, with a global 5-year survival rate <20%. Early metastasis and recurrence remain major challenges for lung cancer treatment. The stemness property of cancer cells has been suggested to play a key role in cancer plasticity, metastasis and drug-resistance, and is a potential target for drug development. In this study, we found that in non-small cell lung cancer (NSCLC), BMI1 and MCL1 play crucial roles of cancer stemness including invasion, chemo-resistance and tumour initiation. JNK signalling serves as a link between oncogenic pathway or genotoxicity to cancer stemness. The activation of JNK, either by mutant EGFR or chemotherapy agent, stabilized BMI1 and MCL1 proteins through suppressing the expression of E3-ubiquitin ligase HUWE1. In lung cancer patient samples, high level of BMI1 is correlated with poor survival, and the expression of BMI1 is positively correlated with MCL1. A novel small-molecule, BI-44, was developed, which effectively suppressed BMI1/MCL1 expressions and inhibited tumour formation and progression in preclinical models. Targeting cancer stemness mediated by BMI1/MCL1 with BI-44 provides the basis for a new therapeutic approach in NSCLC treatment.

Pigment epithelium-derived factor peptide reverses mouse age-related meibomian gland atrophy.

Age-related meibomian gland (MG) atrophy, characterized by decreased meibocyte proliferation, is one of the causes of meibomian gland dysfunction (MGD), which leads to dry eye disease. Currently, there is no available treatment effectively preventing or reversing the decreased cell proliferation and acinar tissue atrophy. In this study, we investigated the therapeutic effects of a pigment epithelium-derived factor (PEDF) peptide in treating this condition. We found abundant expression of PEDF in the nucleus of acinar basal cells, but not in mature meibocytes, and that the expression levels were significantly decreased in the aged mice. We next treated the aged mice (15-month old) with atrophic MGs using a synthetic PEDF-derived peptide 29-mer (residues 93-121). We found that 29-mer effectively stimulated acinar basal cell proliferation and the following mature meibocyte proliferation in the atrophied MGs. In addition, the treatment increased ΔNp63 and Lrig1 expressions in acinar basal cells. Finally, the aged mice receiving the treatment showed MG growth and improved tear film break-up time. In conclusion, the 29-mer treatment is effective in promoting MG acinar basal cell proliferation and enlarging the acinar size of MG, as well as improving MG function in aged mice, suggesting a therapeutic potential of the PEDF-derived short peptide in ameliorating age-related MGD.

Hedgehog pathway maintains cell survival under stress conditions, and drives drug resistance in lung adenocarcinoma.

Hedgehog (HH) pathway plays an important role in embryonic development, but is largely inactive in adult except for tissue repair. Aberrant activation of HH pathway has been found in a variety of cancer types. In non-small cell lung cancer, however, the role and importance of HH pathway remain controversial. In the current study, we found that HH pathway was maintained in low activity in lung adenocarcinoma (LAC) cells under normal culture condition, but was highly induced in response to stress conditions. Activation of HH pathway promoted cell survival, growth, and invasion partially through HGF and MET signaling. Hedgehog-Interacting Protein (HHIP), a cell-surface negative regulator of HH pathway, was epigenetically silenced in LAC. Overexpression of HHIP blocked the activation of HH and HGF/MET pathways, and made cells significantly more susceptible to stress conditions. In LAC cells with acquired resistance to Epidermal Growth Factor Receptor Tyrosin Kinase Inhibitor (EGFR-TKI), we found that a part of tumor cells were much more sensitive to HH or HGF/MET inhibitors, suggesting an oncogenic addiction shift from EGFR to HH and HGF/MET pathways. In conclusion, this study showed that HH pathway is a survival signaling that drives LAC cell growth under stress conditions, and HHIP is a key regulator to block the induction of HH pathway. Targeting the HH pathway through inhibitors or HHIP thus holds promise to address EGFR-TKI resistance in LAC in clinic.

Polyethyleneimine and DNA nanoparticles-based gene therapy for acute lung injury.

Acute lung injury (ALI) is a devastating clinical syndrome causing a substantial mortality, but to date without any effective pharmacological management in clinic. Here, we tested whether nanoparticles based on polyethylenimine (PEI) and DNA could be a potential treatment. In mouse model of ALI induced by lipopolysaccharide (LPS) (10mg/kg), intravenous injection of PEI/DNA mediated a rapid (in 6h) and short-lived transgene expression in lung, with alveolar epithelial cells as major targets. When ß2-Adrenergic Receptor (ß2AR) was applied as therapeutic gene, PEI/ß2AR treatment significantly attenuated the severity of ALI, including alveolar fluid clearance, lung water content, histopathology, bronchioalveolar lavage cellularity, protein concentration, and inflammatory cytokines in mice with pre-existing ALI. In high-dose LPS (40 mg/kg)-induced ALI, post-injury treatment of PEI/ß2AR significantly improved the 5-day survival of mice from 28% to 64%. These data suggest that PEI/DNA nanoparticles could be an effective agent in future clinical application for ALI treatment. FROM THE CLINICAL EDITOR: In this novel study, PEI/DNA nanoparticles are presented as an effective agent for the treatment of the devastating and currently untreatable syndrome of acute lung injury, using a rodent model system.

The PML isoform IV is a negative regulator of nuclear EGFR's transcriptional activity in lung cancer.

Epidermal growth factor receptor (EGFR) is a membrane-bound receptor tyrosine kinase, which can transduce intracellular signals responsible for cell proliferation. It is frequently overexpressed and/or constitutively activated in non-small cell lung cancer and thus is considered as a major cause of this disease. Recently, EGFR has been found in the nucleus where the nuclear EGFR (nEGFR) can function as a transcription factor activating the transcription of genes such as cyclin D1 gene (CCND1), which is essential for cell proliferation. Nevertheless, how nEGFR's transcriptional activity is regulated remains unclear. Promyelocytic leukemia protein (PML) is a tumor suppressor, which is lost in various cancers including lung cancer. However, the role of PML in the suppression of lung cancer growth is still unclear. When we investigated the role of PML in the regulation of lung cancer cell growth, we found that PML isoform IV (PMLIV) preferentially represses the growth of lung cancer cells bearing constitutively active EGFR. Besides, when growing in the EGFR activating conditions, the growth of EGFR wild-type bearing A549 cells has been repressed by PMLIV overexpression. We also found that PMLIV can interact physically with nEGFR and represses the transcription of nEGFR target genes. We showed that PMLIV is recruited by nEGFR to the target promoters and reduces the promoter histone acetylation level via HDAC1. Together, our results suggest that PMLIV interacts with nEGFR upon EGFR activation and represses the transcription of nEGFR target genes such as CCND1 and thus leading to inhibition of the lung cancer cell growth.

Lifelong reporter gene imaging in the lungs of mice following polyethyleneimine-mediated sleeping-beauty transposon delivery.

Polyethyleneimine (PEI) is a cationic polymer that is effective in gene delivery in vivo. Plasmid DNA incorporating the Sleeping-Beauty (SB) transposon has been shown to induce long-term transgene expression in mouse lungs after PEI-mediated delivery. In the current report, we followed the reporter gene expression mediated by PEI/SB delivery in lungs of mice using the non-invasive bioluminescent imaging (BLI) technology. After delivery, the reporter gene signal showed a rapid decay in the first two weeks to a nearly undetectable level, but then the signal augmented gradually in the following weeks and finally reached a stable level that maintained until the natural death of animals. The stabilization of transgene expression is associated with the multiplication of a small number of PEI/SB-labeled alveolar cells, which proliferated both under normal conditions and in response to acute local injury for epithelia repair, and may play a role in long-term homeostatic maintenance in alveoli. The data presented here suggests that systemic delivery of PEI/SB induces stable transfection specifically in a small population of alveolar progenitor cells. The technique provides a promising platform for future research in distal lung biology and tissue regenerative therapy.

Identification of a new peptide for fibrosarcoma tumor targeting and imaging in vivo.

A 12-mer amino acid peptide SATTHYRLQAAN, denominated TK4, was isolated from a phage-display library with fibrosarcoma tumor-binding activity. In vivo biodistribution analysis of TK4-displaying phage showed a significant increased phage titer in implanted tumor up to 10-fold in comparison with normal tissues after systemic administration in mouse. Competition assay confirmed that the binding of TK4-phage to tumor cells depends on the TK4 peptide. Intravenous injection of (131)I-labeled synthetic TK4 peptide in mice showed a tumor retention of 3.3% and 2.7% ID/g at 1- and 4-hour postinjection, respectively. Tumor-to-muscle ratio was 1.1, 5.7, and 3.2 at 1-, 4-, and 24-hour, respectively, and tumors were imaged on a digital γ-camera at 4-hour postinjection. The present data suggest that TK4 holds promise as a lead structure for tumor targeting, and it could be further applied in the development of diagnostic or therapeutic agent.

Poor intercellular transport and absence of enhanced antiproliferative activity after non-viral gene transfer of VP22-P53 or P53-VP22 fusions into p53 null cell lines in vitro or in vivo.

BACKGROUND: The herpes simplex virus type 1 (HSV-1) VP22 protein has the property to mediate intercellular trafficking of heterologous proteins fused to its C- or N-terminus. We have previously shown improved delivery and enhanced therapeutic effect in vitro and in vivo with a P27-VP22 fusion protein. In this report, we were interested in studying the spread and biological activity of VP22 fused to the P53 tumor suppressor. METHODS: Expression of the VP22-P53 and P53-VP22 fusion proteins was shown by Western blot and intercellular spreading was monitored by immunofluorescence on transiently transfected cells. In vitro antiproliferative activity of wild-type (wt) P53 and P53-VP22 was assessed by proliferation assays and transactivating ability was studied by a reporter gene test and a gel-shift assay. Antitumor activity was also tested in vivo by intratumoral injections of naked DNA in a model of subcutaneous tumors implanted in nude mice. RESULTS: Our results show that the C-terminal fusion or the N-terminal P53-VP22 fusion proteins are not able to spread as efficiently as VP22. Moreover, we demonstrate that VP22-P53 does not possess any transactivating ability. P53-VP22 has an antiproliferative activity, but this activity is not superior to the one of P53 alone, in vitro or in vivo. CONCLUSIONS: Our study indicates that a gene transfer strategy using VP22 cannot be considered as a universal system to improve the delivery of any protein.