Eric Van Buren

Differentially expressed miRNAs in cancer-stem-like cells: markers for tumor cell aggressiveness of pancreatic cancer.

Pancreatic cancer (PC) is one of the most deadly cancers. The higher mortality is in part due to treatment resistance and early onset of metastasis. The existence of cancer-stem-like cells (CSLCs) has been widely accepted to be responsible for tumor aggressiveness in PC. Emerging evidence suggests that CSLCs have the capacity for increased cell growth, cell migration/invasion, metastasis, and treatment resistance, which leads to poor clinical outcome. However, the molecular role of CSLCs in tumor development and progression is poorly understood. Therefore, mechanistic understanding, and targeted killing of CSLCs may provide a newer therapeutic strategy for the treatment of PC. It has been well accepted that microRNAs (miRNAs) play critical roles during tumor development and progression through deregulation of multiple genes. Moreover, deregulated expression of miRNAs may also play a key role in the regulation of CSLC characteristics and functions. Here we show that isolated CD44(+)/CD133(+)/EpCAM(+) cells (triple-marker-positive cells) from human PC cell lines, MiaPaCa-2 and L3.6pl cells, display aggressive characteristics, such as increased cell growth, clonogenicity, cell migration, and self-renewal capacity, which is consistent with overexpression of CSLC signatures/markers. We also found deregulated expression of over 400 miRNAs, including let-7, miR-30, miR-125b, and miR-335, in CSLCs. As a proof-of-concept, knockdown of miR-125b resulted in the inhibition of tumor cell aggressiveness of CSLCs (triple-marker-positive cells), consistent with the downregulation of CD44, EpCAM, EZH2, and snail. These results clearly suggest the importance of miRNAs in the regulation of CSLC characteristics, and may serve as novel targets for therapy.

Combining anticancer agents photodynamic therapy and LCL85 leads to distinct changes in the sphingolipid profile, autophagy, caspase-3 activation in the absence of cell death, and long-term sensitization.

Two anticancer agents, LCL85 and photodynamic therapy (PDT) were combined to test whether the combination PDT/LCL85 evokes changes in the sphingolipid (SL) profile and promotes cell death. Treatment of SCCVII mouse squamous carcinoma cells using the silicone phthalocyanine Pc 4 for PDT induced increases in the prodeath global ceramides/dihydroceramides (DHceramides), and no changes in the prosurvival sphingosine-1-phosphate (S1P). In contrast, after LCL85, the levels of most ceramides and DHceramides were reduced, whereas the levels of S1P were increased. After PDT/LCL85 the levels of global ceramides and DHceramides, and of S1P, were restored to resting levels. PDT/LCL85 also enhanced the levels of C18-, C20-, and C20:1-ceramide, and C18-DHceramide. Treatment with PDT, with or without LCL85, led to substantial reductions in sphingosine levels. PDT/LCL85 induced enhanced autophagy and caspase-3 activation. None of the treatments affected short-term viability of cells. In contrast, long-term clonogenic survival was reduced not only after PDT or LCL85, but even more after PDT/LCL85. Overall, our data show that short-term exposure to PDT/LCL85 led to distinct signature effects on the SL profile, enhanced autophagy, and caspase-3 activation without cell death. Long-term exposure to PDT/LCL85 enhanced overall cell killing, supporting translational potential of PDT/LCL85.

C16-Ceramide Analog Combined with Pc 4 Photodynamic Therapy Evokes Enhanced Total Ceramide Accumulation, Promotion of DEVDase Activation in the Absence of Apoptosis, and Augmented Overall Cell Killing

Because of the failure of single modality approaches, combination therapy for cancer treatment is a promising alternative. Sphingolipid analogs, with or without anticancer drugs, can improve tumor response. C16-pyridinium ceramide analog LCL30, was used in combination with photodynamic therapy (PDT), an anticancer treatment modality, to test the hypothesis that the combined treatment will trigger changes in the sphingolipid profile and promote cell death. Using SCCVII mouse squamous carcinoma cells, and the silicone phthalocyanine Pc 4 for PDT, we showed that combining PDT with LCL30 (PDT/LCL30) was more effective than individual treatments in raising global ceramide levels, as well as in reducing dihydrosphingosine levels. Unlike LCL30, PDT, alone or combined, increased total dihydroceramide levels. Sphingosine levels were unaffected by LCL30, but were abolished after PDT or the combination. LCL30-triggered rise in sphingosine-1-phosphate was reversed post-PDT or the combination. DEVDase activation was evoked after PDT or LCL30, and was promoted post- PDT/LCL30. Neither mitochondrial depolarization nor apoptosis were observed after any of the treatments. Notably, treatment with the combination resulted in augmented overall cell killing. Our data demonstrate that treatment with PDT/LCL30 leads to enhanced global ceramide levels and DEVDase activation in the absence of apoptosis, and promotion of total cell killing.

Role of cancer stem cells in racial disparity in colorectal cancer.

Although African‐Americans (AAs) have a higher incidence of colorectal cancer (CRC) than White people, the underlying biochemical mechanisms for this increase are poorly understood. The current investigation was undertaken to examine whether differences in self‐renewing cancer stem/stem‐like cells (CSCs) in the colonic mucosa, whose stemness is regulated by certain microRNAs (miRs), could partly be responsible for the racial disparity in CRC. The study contains 53 AAs and 47 White people. We found the number of adenomas and the proportion of CD44+CD166− CSC phenotype in the colon to be significantly higher in AAs than White people. MicroRNAs profile in CSC‐enriched colonic mucosal cells, expressed as ratio of high‐risk (≥3 adenomas) to low‐risk (no adenoma) CRC patients revealed an 8‐fold increase in miR‐1207‐5p in AAs, compared to a 1.2‐fold increase of the same in White people. This increase in AA was associated with a marked rise in lncRNA PVT1 (plasmacytoma variant translocation 1), a host gene of miR‐1207‐5p. Forced expression of miR‐1207‐5p in normal human colonic epithelial cells HCoEpiC and CCD841 produced an increase in stemness, as evidenced by morphologically elongated epithelial mesenchymal transition( EMT) phenotype and significant increases in CSC markers (CD44, CD166, and CD133) as well as TGF‐β, CTNNB1, MMP2, Slug, Snail, and Vimentin, and reduction in Twist and N‐Cadherin. Our findings suggest that an increase in CSCs, specifically the CD44+CD166− phenotype in the colon could be a predisposing factor for the increased incidence of CRC among AAs. MicroRNA 1207‐5p appears to play a crucial role in regulating stemness in colonic epithelial cells in AAs.

Increased killing of SCCVII squamous cell carcinoma cells after the combination of Pc 4 photodynamic therapy and dasatinib is associated with enhanced caspase-3 activity and ceramide synthase 1 upregulation

Photodynamic therapy (PDT) is not always effective as an anticancer treatment, therefore, PDT is combined with other anticancer agents for improved efficacy. The combination of dasatinib and PDT with the silicone phthalocyanine photosensitizer Pc 4 was assessed for increased killing of SCCVII mouse squamous cell carcinoma cells, a preclinical model of head and neck squamous cell carcinoma, using apoptotic markers and colony formation as experimental end-points. Because each of these treatments regulates the metabolism of the sphingolipid ceramide, their effects on mRNA levels of ceramide synthase, a ceramide-producing enzyme, and the sphingolipid profile were determined. PDT + dasatinib induced an additive loss of clonogenicity. Unlike PDT alone or PDT + dasatinib, dasatinib induced zVAD-fmk-dependent cell killing. PDT or dasatinib-induced caspase-3 activation was potentiated after the combination. PDT alone induced mitochondrial depolarization, and the effect was inhibited after the combination. Annexin V+ and propidium iodide+ cells remained at control levels after treatments. In contrast to PDT alone, dasatinib induced upregulation of ceramide synthase 1 mRNA, and the effect was enhanced after the combination. Dasatinib induced a modest increase in C20:1-and C22-ceramide but had no effect on total ceramide levels. PDT increased the levels of 12 individual ceramides and total ceramides, and the addition of dasatinib did not affect these increases. PDT alone decreased substantially sphingosine levels and inhibited the activity of acid ceramidase, an enzyme that converts ceramide to sphingosine. The data suggest that PDT-induced increases in ceramide levels do not correlate with ceramide synthase mRNA levels but rather with inhibition of ceramidase. Cell killing was zVAD-fmk-sensitive after dasatinib but not after either PDT or the combination and enhanced cell killing after the combination correlated with potentiated caspase-3 activation and upregulation of ceramide synthase 1 mRNA but not the production of ceramide. The data imply potential significance of the combination for cancer treatment.

Maspin Expression in Prostate Tumor Cells Averts Stemness and Stratifies Drug Sensitivity

Future curative cancer chemotherapies have to overcome tumor cell heterogeneity and plasticity. To test the hypothesis that the tumor suppressor maspin may reduce microenvironment-dependent prostate tumor cell plasticity and thereby modulate drug sensitivity, we established a new schematic combination of two-dimensional (2D), three-dimensional (3D), and suspension cultures to enrich prostate cancer cell subpopulations with distinct differentiation potentials. We report here that depending on the level of maspin expression, tumor cells in suspension and 3D collagen I manifest the phenotypes of stem-like and dormant tumor cell populations, respectively. In suspension, the surviving maspin-expressing tumor cells lost the self-renewal capacity, underwent senescence, lost the ability to dedifferentiate in vitro, and failed to generate tumors in vivo. Maspin-nonexpressing tumor cells that survived the suspension culture in compact tumorspheres displayed a higher level of stem cell marker expression, maintained the self-renewal capacity, formed tumorspheres in 3D matrices in vitro, and were tumorigenic in vivo. The drug sensitivities of the distinct cell subpopulations depend on the drug target and the differentiation state of the cells. In 2D, docetaxel, MS275, and salinomycin were all cytotoxic. In suspension, while MS275 and salinomycin were toxic, docetaxel showed no effect. Interestingly, cells adapted to 3D collagen I were only responsive to salinomycin. Maspin expression correlated with higher sensitivity to MS275 in both 2D and suspension and to salinomycin in 2D and 3D collagen I. Our data suggest that maspin reduces prostate tumor cell plasticity and enhances tumor sensitivity to salinomycin, which may hold promise in overcoming tumor cell heterogeneity and plasticity.

Enhanced apoptotic cancer cell killing after Foscan photodynamic therapy combined with fenretinide via de novo sphingolipid biosynthesis pathway

We and others have shown that stresses, including photodynamic therapy (PDT), can disrupt the de novo sphingolipid biosynthesis pathway, leading to changes in the levels of sphingolipids, and subsequently, modulation of cell death. The de novo sphingolipid biosynthesis pathway includes a ceramide synthase-dependent reaction, giving rise to dihydroceramide, which is then converted in a desaturase-dependent reaction to ceramide. In this study we tested the hypothesis that combining Foscan-mediated PDT with desaturase inhibitor fenretinide (HPR) enhances cancer cell killing. We discovered that by subjecting SCC19 cells, a human head and neck squamous cell carcinoma cell line, to PDT+HPR resulted in enhanced accumulation of C16-dihydroceramide, not ceramide. Concomitantly, mitochondrial depolarization was enhanced by the combined treatment. Enhanced activation of caspase-3 after PDT+HPR was inhibited by FB. Enhanced clonogenic cell death after the combination was sensitive to FB, as well as Bcl2- and caspase inhibitors. Treatment of mouse SCCVII squamous cell carcinoma tumors with PDT+HPR resulted in improved long-term tumor cures. Overall, our data showed that combining PDT with HPR enhanced apoptotic cancer cell killing and antitumor efficacy of PDT. The data suggest the involvement of the de novo sphingolipid biosynthesis pathway in enhanced apoptotic cell killing after PDT+HPR, and identify the combination as a novel more effective anticancer treatment than either treatment alone.