Increasing evidence shows that hepatocellular carcinoma (HCC) is a principal cause of cancer-related mortality globally, especially among Asian and African populations. Collagen type I α1 (COL1A1) is the major component of type I collagen. While aberrant expression of COL1A1 and COL1A2 is implicated in numerous cancers, the differential role of COL1A1 in malignant, premalignant and normal tissues remains unclear, and its clinical significance in HCC has not been elucidated. In this study, using bioinformatics analysis of publicly-available HCC microarray data from Gene Expression Omnibus (GEO) and RNAseq data from The Cancer Genome Atlas (TCGA) database, we determined that COL1A1 is significantly upregulated in HCC tumor tissues in comparison to normal tissues. Our analysis also revealed that COL1A1 confers survival advantage and enhanced oncogenicity on HCC cells. Interestingly, the siRNA-mediated silencing of COL1A1 expression (siCOLIA1) suppressed HCC cells clonogenicity, motility, invasiveness and tumorsphere formation. Concomitantly, siCOL1A1 abrogated Slug-dependent epithelial-to-mesenchymal transition (EMT) and HCC stemness gene-signature, by attenuating expression of stemness markers SOX2, OCT4 and CD133. The present study provides some mechanistic insight into COL1A1 activity in HCC and highlights its putative role as an important diagnostic biomarker and potential therapeutic target in early development and metastasis of HCC.

Rationale: Cancer stem cell (CSC) theory has drawn much attention, with evidence supporting the contribution of stem cells to tumor initiation, relapse, and therapy resistance. Objectives: To screen drugs that target CSCs to improve the current treatment outcome and overcome drug resistance in patients with lung cancer. Methods: We used publicly available embryonic stem cell and CSC-associated gene signatures to query the Connectivity Map for potential drugs that can, at least in part, reverse the gene expression profile of CSCs. High scores were noted for several phenothiazine-like antipsychotic drugs, including trifluoperazine. We then treated lung CSCs with different EGFR mutation status with trifluoperazine to examine its anti-CSC properties. Lung CSCs resistant to epidermal growth factor receptor–tyrosine kinase inhibitor or cisplatin were treated with trifluoperazine plus gefitinib or trifluoperazine plus cisplatin. Animal models were used for in vivo validation of the anti-CSC effect and synergistic effect of trifluoperazine with gefitinib. Measurements and Main Results: We demonstrated that trifluoperazine inhibited CSC tumor spheroid formation and down-regulated the expression of CSC markers (CD44/CD133). Trifluoperazine inhibited Wnt/β-catenin signaling in gefitinib-resistant lung cancer spheroids. The combination of trifluoperazine with either gefitinib or cisplatin overcame drug resistance in lung CSCs. Trifluoperazine inhibited the tumor growth and enhanced the inhibitory activity of gefitinib in lung cancer metastatic and orthotopic CSC animal models. Conclusions: Using in silico drug screening by Connectivity Map followed by empirical validations, we repurposed an existing phenothiazine-like antipsychotic drug, trifluoperazine, as a potential anti-CSC agent that could overcome epidermal growth factor receptor–tyrosine kinase inhibitor and chemotherapy resistance.

Platinum-based chemotherapy is the first-line treatment for non–small cell lung cancer, but recurrence occurs in most patients. Recent evidence suggests that CD133+ cells are the cause of drug resistance and tumor recurrence. However, the correlation between chemotherapy and regulation of CD133+ cells has not been investigated methodically. In this study, we revealed that CD133+ lung cancer cells labeled by a human CD133 promoter–driven GFP reporter exhibited drug resistance and stem cell characteristics. Treatment of H460 and H661 cell lines with low-dose cisplatin (IC20) was sufficient to enrich CD133+ cells, to induce DNA damage responses, and to upregulate ABCG2 and ABCB1 expression, which therefore increased the cross-resistance to doxorubicin and paclitaxel. This cisplatin-induced enrichment of CD133+ cells was mediated through Notch signaling as judged by increased levels of cleaved Notch1 (NICD1). Pretreatment with the γ-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester (DAPT), or Notch1 short hairpin RNAs (shRNA) remarkably reduced the cisplatin-induced enrichment of CD133+ cells and increased the sensitivity to doxorubicin and paclitaxel. Ectopic expression of NICD1 reversed the action of DAPT on drug sensitivity. Immunohistochemistry showed that CD133+ cells were significantly increased in the relapsed tumors in three of six patients with lung cancer who have received cisplatin treatment. A similar effect was observed in animal experiments as cisplatin treatment increased Notch1 cleavage and the ratio of CD133+ cells in engrafted tumors. Intratumoral injection of DAPT with cisplatin treatment significantly reduced CD133+ cell number. Together, our results showed that cisplatin induces the enrichment of CD133+ cells, leading to multidrug resistance by the activation of Notch signaling. Cancer Res; 73(1); 406–16. ©2012 AACR.

Background: Recently, we demonstrated that Astragalus polysaccharide (PG2), the active ingredient in dried roots of astragalus membranaceus, ameliorates cancer symptom clusters and improves quality of life (QoL) in patients with metastatic disease by modulating inflammatory cascade against the background roles of inflammatory cells, including macrophages, dendritic cells (DCs), and cytotoxic T lymphocytes (CTLs) in tumor initiation, metastasis, and progression. Nevertheless, the role of PG2 in the modulation of anticancer immunogenicity and therapeutic response remains relatively underexplored and unclear. Purpose: The present study investigates how and to what extent PG2 modulates cellular and biochemical components of the inflammatory cascade and enhances anticancer immunity, as well as the therapeutic implication of these bio-events in patients with lung cancer. Methods and Results: Herein, we demonstrated that PG2 significantly increased the M1/M2 macrophage polarization ratio in non-small cell carcinoma (NSCLC) H441 and H1299 cells. This PG2-induced preferential pharmacologic up-regulation of tumoral M1 population in vitro positively correlated with the downregulation of tumor-promoting IL-6 and IL-10 expression in NSCLC cell-conditioned medium, with concomitant marked inhibition of cell proliferation, clonogenicity, and tumorsphere formation. Our ex vivo results, using clinical sample from our NSCLC cohort, demonstrated that PG2 also promoted the functional maturation of DCs with consequent enhancement of T cell-mediated anticancer immune responses. Consistent with the in vitro and ex vivo results, our in vivo studies showed that treatment with PG2 elicited significant time-dependent depletion of the tumor-associated M2 population, synergistically enhanced the anti-M2-based anticancer effect of cisplatin, and inhibited xenograft tumor growth in the NSCLC mice models. Moreover, in the presence of PG2, cisplatin-associated dyscrasia and weight-loss was markedly suppressed. Conclusion: These results do indicate a therapeutically-relevant role for PG2 in modulating the M1/M2 macrophage pool, facilitating DC maturation and synergistically enhancing the anticancer effect of conventional chemotherapeutic agent, cisplatin, thus laying the foundation for further exploration of the curative relevance of PG2 as surrogate immunotherapy and/or clinical feasibility of its use for maintenance therapy in patients with lung cancer.

We report a novel alkaline extractable protein of the sperm head that exclusively resides in the post-acrosomal sheath region of the perinuclear theca (PT) and is expressed and assembled in elongating spermatids. It is a protein that shares sequence homology to the N-terminal half of WW domain-binding protein 2, while the C-terminal half is unique and rich in proline. A functional PPXY consensus binding site for group-I WW domain-containing proteins, and numerous unique repeating motifs, YGXPPXG, are identified in the proline-rich region. Considering these molecular characteristics, we designated this protein PAWP for postacrosomal sheath WW domain-binding protein. Microinjection of recombinant PAWP or alkaline PT extract into metaphase II-arrested porcine, bovine, macaque, and Xenopus oocytes induced a high rate of pronuclear formation, which was prevented by co-injection of a competitive PPXY motif containing peptide derived from PAWP but not by co-injection of the point-mutated peptide. Intracytoplasmic sperm injection (ICSI) of porcine oocytes combined with co-injection of the competitive PPXY peptide or an anti-recombinant PAWP antiserum prevented pronuclear formation and arrested fertilization. Conversely, co-injection of the modified PPXY peptide, when the tyrosine residue of PPXY was either phosphorylated or substituted with phenylalanine, did not prevent ICSI-induced fertilization. This study uncovers a group I WW domain module signal transduction event within the fertilized egg that appears compulsory for meiotic resumption and pronuclear development during egg activation and provides compelling evidence that a PPXY motif of sperm-contributed PAWP can trigger these events.