Oncogenic Impact of TONSL, a Homologous Recombination Repair Protein at the Replication Fork, in Cancer Stem Cells.

We investigated the role of TONSL, a mediator of homologous recombination repair (HRR), in stalled replication fork double-strand breaks (DSBs) in cancer. Publicly available clinical data (tumors from the ovary, breast, stomach and lung) were analyzed through KM Plotter, cBioPortal and Qomics. Cancer stem cell (CSC)-enriched cultures and bulk/general mixed cell cultures (BCCs) with RNAi were employed to determine the effect of TONSL loss in cancer cell lines from the ovary, breast, stomach, lung, colon and brain. Limited dilution assays and ALDH assays were used to quantify the loss of CSCs. Western blotting and cell-based homologous recombination assays were used to identify DNA damage derived from TONSL loss. TONSL was expressed at higher levels in cancer tissues than in normal tissues, and higher expression was an unfavorable prognostic marker for lung, stomach, breast and ovarian cancers. Higher expression of TONSL is partly associated with the coamplification of TONSL and MYC, suggesting its oncogenic role. The suppression of TONSL using RNAi revealed that it is required in the survival of CSCs in cancer cells, while BCCs could frequently survive without TONSL. TONSL dependency occurs through accumulated DNA damage-induced senescence and apoptosis in TONSL-suppressed CSCs. The expression of several other major mediators of HRR was also associated with worse prognosis, whereas the expression of error-prone nonhomologous end joining molecules was associated with better survival in lung adenocarcinoma. Collectively, these results suggest that TONSL-mediated HRR at the replication fork is critical for CSC survival; targeting TONSL may lead to the effective eradication of CSCs.

Clinical and biochemical relevance of monounsaturated fatty acid metabolism targeting strategy for cancer stem cell elimination in colon cancer.

Lipid metabolism is associated with colon cancer prognosis and incidence. Stearoyl-CoA desaturase 1 (SCD1), which converts fully saturated fatty acids (SFAs) to monounsaturated fatty acids (MUFAs), has been suggested as a vulnerable target for selective elimination of cancer stem cells (CSCs). However, the clinical significance and physiological role of SCD1 in CSCs has not been well demonstrated. Here, we showed the clinical and biochemical relevance of blocking SCD1 to target CSCs by analyzing human colon cancer data from TCGA and through lipidomic profiling of CSCs with or without SCD1 inhibition using mass spectrometry. Positive associations between SCD1 expression and colorectal cancer patient clinical status and the expression of CSC-related genes (WNT and NOTCH signaling) were found based on TCGA data analysis. Lipidomic profiling of CSCs and bulk cancer cells (BCCs) using mass spectrometry revealed that colon CSCs contained a distinctive lipid profile, with higher free MUFA and lower free SFA levels than in BCCs, suggesting that enhanced SCD1 activity generates MUFAs that may support WNT signaling in CSCs. In addition, all identified phosphatidyl-ethanolamine-containing MUFAs were found at higher levels in CSCs. Interestingly, we observed lower phosphatidyl-serine (18:1/18:0), phosphatidyl-choline (PC; p-18:0/18:1)), and sphingomyelin (SM; d18:1/20:0 or d16:1/22:0) levels in CSCs than in BCCs. Of those, SCD1 inhibition, which efficiently diminished free MUFA levels, increased those specific PC and SM and MUFAs in CSCs promptly. These results suggest that these specific lipid composition is critical for CSC stem cell maintenance. In addition, not only free MUFAs, which are known to be required for WNT signaling, but also other phospholipids, such as SM, which are important for lipid raft formation, may mediate other cell signaling pathways that support CSC maintenance. Comparison of the lipidomic profiles of colon cancer cells with those of previously reported for glioma cells further demonstrated the tissue specific characteristics of lipid metabolism in CSCs.

Evodiamine Eliminates Colon Cancer Stem Cells via Suppressing Notch and Wnt Signaling.

Evodiamine, an alkaloid contained in traditional Asian herbal medicines that have been used for hundreds years, is interesting due to its cytotoxic effects against many cancers. We examined the effect of evodiamine on the cancer stem cell (CSC) population and the bulk cultured cancer cells (BCC) of colon cancers to examine the double targeting effect. We found that three colon cancer cell lines' BCC and CSC are effectively targeted by evodiamine. Evodiamine was able to suppress BCC proliferation and induce apoptosis of the cells captured in G2/M phase, as previously reported. However, evodiamine did not cause the accumulation of CSCs at a certain stage of the cell cycle, resulting in the elimination of stemness through an unknown mechanism. By analyzing the expression of 84 genes related to CSCs in two colon cancer cell lines' CSC, as well as performing further informatics analyses, and quantitative RT-PCR analyses of 24 CSC genes, we found that evodiamine suppressed the expression of the genes that control key signaling pathways of CSC, namely, WNT and NOTCH signaling, to lead CSC elimination. These results suggest that evodiamine should be further developed for targeting both BCCs and CSCs in colon cancers.

Loss-of-function screens of druggable targetome against cancer stem-like cells.

Cancer stem-like cells (CSLCs) contribute to the initiation and recurrence of tumors and to their resistance to conventional therapies. In this study, small interfering RNA (siRNA)-based screening of ∼4800 druggable genes in 3-dimensional CSLC cultures in comparison to 2-dimensional bulk cultures of U87 glioma cells revealed 3 groups of genes essential for the following: survival of the CSLC population only, bulk-cultured population only, or both populations. While diverse biologic processes were associated with siRNAs reducing the bulk-cultured population, CSLC-eliminating siRNAs were enriched in a few functional categories, such as lipid metabolism, protein metabolism, and gene expression. Interestingly, siRNAs that selectively reduced CSLC only were found to target genes for cholesterol and unsaturated fatty acid synthesis. The lipidomic profile of CSLCs revealed increased levels of monounsaturated lipids. Pharmacologic blockage of these target pathways reduced CSLCs, and this effect was eliminated by addition of downstream metabolite products. The present CSLC-sensitive target categories provide a useful resource that can be exploited for the selective elimination of CSLCs.-Song, M., Lee, H., Nam, M.-H., Jeong, E., Kim, S., Hong, Y., Kim, N., Yim, H. Y., Yoo, Y.-J., Kim, J. S., Kim, J.-S., Cho, Y.-Y., Mills, G. B., Kim, W.-Y., Yoon, S. Loss-of-function screens of druggable targetome against cancer stem-like cells.

Selective Targeting of Cancer Stem Cells by 2-Aminodihydroquinoline Analogs.

Many aminodihydroquinoline compounds have been studied to determine their cytotoxicity to cancer cells. However, anti-cancer stem cells (CSCs) activity of aminodihydroquinoline has not been tested in spite that CSC is believed to do an important roles in chemotherapy resistance and recurrence. The CSC selective targeting activities of 10 recently synthesized 2-aminodihydroquinoline analogs were examined on CSCs and bulk culture of a glioblastoma cell line. A diethylaminopropyl substituted aminodihydroquinoline, 5h, showed a strong anti-CSC effect and general cytotoxicity. However, a benzyl substituted aminodihydroquinoline, 5i, displayed the most effective anti-CSC effect, with no or small significant cytotoxic effect in bulk culture conditions. While 5h temporarily enhanced CSC marker-positive cells and eventually suppressed the CSC population, which is similar to other cytotoxic anticancer reagents reported, 5i selectively eliminated CSC marker-positive cells based on fluorescence activated cell sorter (FACS) analysis. 5h also temporarily activated some genes associated with signaling required for CSC, while 5i selectively suppressed these genes supporting that the differential effects are resulted from different molecular responses. In addition, the selective CSC effect is also found against a colon cancer cell line. Collectively, we suggest that these two novel aminodihydroquinoline compounds possess novel anti-CSC effects in colon and brain tumor derived cell lines probably through independent pathways.

Evodiamine selectively targets cancer stem-like cells through the p53-p21-Rb pathway.

In spite of the recent improvements, the resistance to chemotherapy/radiotherapy followed by relapse is the main hurdle for the successful treatment of breast cancer, a leading cause of death in women. A small population of breast cancer cells that have stem-like characteristics (cancer stem-like cells; CSLC) may contribute to this resistance and relapse. Here, we report on a component of a traditional Chinese medicine, evodiamine, which selectively targets CSLC of breast cancer cell lines MCF7 and MDAMB 231 at a concentration that does show a little or no cytotoxic effect on bulk cancer cells. While evodiamine caused the accumulation of bulk cancer cells at the G2/M phase, it did not hold CSLC in a specific cell cycle phase but instead, selectively killed CSLC. This was not due to the culture of CSLC in suspension or without FBS. A proteomic analysis and western blotting revealed that evodiamine changed the expression of cell cycle regulating molecules more efficiently in CSLC cells than in bulk cancer cells. Surprisingly, evodiamine selectively activated p53 and p21 and decreased inactive Rb, the master molecules in G1/S checkpoint. These data collectively suggest a novel mechanism involving CSLC-specific targeting by evodiamine and its possible use to the therapy of breast cancer.

Kazinol-E is a specific inhibitor of ERK that suppresses the enrichment of a breast cancer stem-like cell population.

Growing evidence shows that cancer stem-like cells (CSLCs) contribute to breast cancer recurrence and to its resistance to conventional therapies. The extracellular signal-regulated kinase (ERK) signaling pathway is a major determinant in the control of diverse cellular processes, including the maintenance of CSLCs. In this study, we found that Kazinol-E, an antioxidant flavan from Broussonetia kazinoki, decreased the CSLC population of a breast cancer cell line, MCF7. The CSLC population, characterized by CD44 high/CD24 low expression or by high Aldehyde dehydrogenase 1 activity, was decreased by a concentration of Kazinol-E that did not affect the growth of bulk-cultured MCF7 cells. Kazinol-E did not decrease EGF-induced ERK phosphorylation in CSLCs, but did block the phosphorylation of an ERK substrate, p90RSK2, at Thr359/Ser363. We further demonstrated that EGF-induced ERK activity was blocked by Kazinol-E in a wild-type K-Ras-expressing non-small cell lung cancer cell line H226B. An in vitro kinase assay with purified ERK1 and p90RSK2 as its substrate demonstrated a direct inhibition of ERK activity by Kazinol E. Additionally, a the molecular docking study provided putative binding modes of Kazinol-E into the ATP binding pocket of ERK1 Collectively, these results suggest that Kazinol-E is a direct inhibitor of ERK1, and more studies are warranted to develop this reagent for therapeutic breast CSLC targeting.

Role of circulating mitochondria in venous thrombosis in glioblastoma.

BACKGROUND: Many patients with glioblastoma multiforme (GBM) develop deep venous thrombosis or pulmonary emboli. Cell-free circulating mitochondria increase after brain injury and are associated with coagulopathy. OBJECTIVES: This study evaluated whether mitochondria play a role in the GBM-induced hypercoagulable state. METHODS: We examined the correlation between cell-free circulating mitochondria and venous thrombosis in patients with GBM and the impact of mitochondria on venous thrombosis in mice with inferior vena cava stenosis. RESULTS: Using plasma samples of 82 patients with GBM, we found that patients with GBM had a higher number of mitochondria in their plasma (GBM with venous thromboembolism [VTE],: 2.8 × 107 mitochondria/mL; GBM without VTE, 1.9 × 107 mitochondria/mL) than that in healthy control subjects (n = 17) (0.3 × 107 mitochondria/mL). Interestingly, patients with GBM and VTE (n = 41) had a higher mitochondria concentration than patients with GBM without VTE (n = 41). In a murine model of inferior vena cava stenosis, intravenous delivery of mitochondria resulted in an increased rate of venous thrombosis compared with that in controls (70% and 28%, respectively). Mitochondria-induced venous thrombi were neutrophil-rich and contained more platelets than those in control thrombi. Furthermore, as mitochondria are the only source of cardiolipin in circulation, we compared the concentration of anticardiolipin immunoglobulin G in plasma samples of patients with GBM and found a higher concentration in patients with VTE (optical density, 0.69 ± 0.04) than in those without VTE (optical density, 0.51 ± 0.04). CONCLUSION: We concluded that mitochondria might play a role in the GBM-induced hypercoagulable state. We propose that quantifying circulating mitochondria or anticardiolipin antibody concentrations in patients with GBM might identify patients at increased risk of VTE.

ARL2 is required for homologous recombination repair and colon cancer stem cell survival.

ARL2 regulates the dynamics of cytological components and is highly expressed in colon cancer tissues. Here, we report novel roles of ARL2 in the cell nucleus and colon cancer stem cells (CSCs). ARL2 is expressed at relatively low levels in K-RAS active colon cancer cells, but its expression is induced in CSCs. Depletion of ARL2 results in M phase arrest exclusively in non-CSC cultured cells; in addition, DNA break stress accumulates in CSCs leading to apoptosis. ARL2 expression is positively associated with the expression of all six RAD51 family genes, which are essential for homologous recombination repair (HRR). Furthermore, ARL2 is required for HRR and detected within chromatin compartments. These results demonstrate the requirement of ARL2 in colon CSC maintenance, which possibly occurs through mediating double-strand break DNA repair in the nucleus.

Platelets Increase the Expression of PD-L1 in Ovarian Cancer.

The interactions between platelets and cancer cells activate platelets and enhance tumor growth. Platelets increase proliferation and epithelial-mesenchymal transition in cancer cells, inhibit anoikis, enhance the extravasation of cancer cells, and protect circulating tumor cells against natural killer cells. Here, we have identified another mechanism by which platelets dampen the immune attack on cancer cells. We found that platelets can blunt the antitumor immune response by increasing the expression of inhibitory immune checkpoint (PD-L1) on ovarian cancer cells in vitro and in vivo. Platelets increased PD-L1 in cancer cells via contact-dependent (through NF-κB signaling) and contact-independent (through TFGßR1/Smad signaling) pathways. Inhibition of NF-κB or TGFßR1 signaling in ovarian cancer cells abrogated platelet-induced PD-L1 expression. Reducing platelet counts or inhibiting platelet functions reduced the expression of PD-L1 in ovarian cancer. On the other hand, an increase in platelet counts increased the expression of PD-L1 in tumor-bearing mice.

Targeting a Lipid Desaturation Enzyme, SCD1, Selectively Eliminates Colon Cancer Stem Cells through the Suppression of Wnt and NOTCH Signaling.

The elimination of the cancer stem cell (CSC) population may be required to achieve better outcomes of cancer therapy. We evaluated stearoyl-CoA desaturase 1 (SCD1) as a novel target for CSC-selective elimination in colon cancer. CSCs expressed more SCD1 than bulk cultured cells (BCCs), and blocking SCD1 expression or function revealed an essential role for SCD1 in the survival of CSCs, but not BCCs. The CSC potential selectively decreased after treatment with the SCD1 inhibitor in vitro and in vivo. The CSC-selective suppression was mediated through the induction of apoptosis. The mechanism leading to selective CSC death was investigated by performing a quantitative RT-PCR analysis of 14 CSC-specific signaling and marker genes after 24 and 48 h of treatment with two concentrations of an inhibitor. The decrease in the expression of Notch1 and AXIN2 preceded changes in the expression of all other genes, at 24 h of treatment in a dose-dependent manner, followed by the downregulation of most Wnt- and NOTCH-signaling genes. Collectively, we showed that not only Wnt but also NOTCH signaling is a primary target of suppression by SCD1 inhibition in CSCs, suggesting the possibility of targeting SCD1 against colon cancer in clinical settings.

The effect of platelet G proteins on platelet extravasation and tumor growth in the murine model of ovarian cancer.

We and other investigators have shown that platelets promote metastasis and the growth of tumors. Our rationale for conducting this study is that platelets' prometastatic and progrowth effects depend on a close encounter between platelets and cancer cells. This interaction occurs inside blood vessels with circulating tumor cells and outside blood vessels with cancer cells residing in the tumor parenchyma. Our hypothesis was that platelet extravasation is required for the effect of platelets on tumor growth. Platelets respond to environmental stimuli by activation of G protein-coupled receptors on their surface. We investigated the impact of various platelet G proteins on the growth of ovarian cancer tumors and platelet extravasation. We used mice with platelet-specific deficiency of Gαi2 (Gi), Gα13 (G13), or Gαq (Gq) in a syngeneic ovarian cancer model. We measured the total weight of tumor nodules resected from tumor-bearing mice. We developed methods for automated whole-slide image acquisition and unbiased computerized image analysis to quantify extravasated platelets. We compared the number of platelets inside tumor nodules of platelet G protein-deficient tumor-bearing mice. We found that deficiency of Gi and G13, but not Gq, in platelets resulted in smaller tumors compared with those in corresponding littermates. Deficiency of Gi and G13 in platelets reduced the number of extravasated platelets by >90%, but deficiency of Gq did not reduce the number of extravasated platelets significantly. The lack of Gi or G13 in platelets reduced platelet extravasation into the tumor and tumor growth.

Ginsenoside Rg3 upregulates myotube formation and mitochondrial function, thereby protecting myotube atrophy induced by tumor necrosis factor-alpha.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginsenoside Rg3 from Panax ginseng has reported to have multiple pharmacological activities including anti-diabetics, anti-inflammation and anti-cancer. However, the effect of ginsenoside Rg3 on myogenic differentiation and muscle atrophy is unknown. AIM TO THE STUDY: In this study, we investigated the myogenic effect and underlying molecular mechanisms of ginsenoside Rg3 on myotube atrophy induced by tumor necrosis factor-α (TNF-α). MATERIALS AND METHODS: C2C12 myoblasts were induced to differentiate for one day followed by the treatment of TNF-α along with vehicle or ginsenoside Rg3 for additional 2 days and subjected to immunoblotting, immunocytochemistry, quantitative RT-PCR and biochemical analysis for mitochondrial function. RESULTS: Ginsenoside Rg3 promotes myogenic differentiation and multinucleated myotube formation through Akt activation in a dose-dependent manner, without any cytotoxicity. Ginsenoside Rg3 treatment restores myotube formation and increases myotube diameters under TNF-α-treated conditions. Ginsenoside Rg3 enhances Akt/mTOR (mammalian target of rapamycin) signaling that in turn stimulates muscle-specific gene expression such as myosin heavy chain (MHC) and Myogenin, and suppresses the expression of muscle-specific ubiquitin ligases. In addition, ginsenoside Rg3 in TNF-α-treated myotubes significantly inhibits the production of mitochondrial ROS and restores mitochondrial membrane potential (MMP) and ATP contents. Furthermore, ginsenoside Rg3 upregulates the activities and expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) and the mitochondrial biogenetic transcription factors, nuclear respiratory factor-1 (NRF1) and mitochondrial transcription factor A (Tfam) in TNF-α-induced myotube atrophy. CONCLUSIONS: This study provides a mechanistic insight into the effect of ginsenoside Rg3 on myogenic differentiation and myotube atrophy, suggesting that ginsenoside Rg3 has a promising potential as a therapeutic or neutraceutical remedy to intervene muscle weakness and atrophy.

ß-catenin/TCF activity regulates IGF-1R tyrosine kinase inhibitor sensitivity in colon cancer.

The availability of large-scale drug screening data on cell line panels provides a unique opportunity to identify predictive biomarkers for targeted drug efficacy. Analysis of diverse drug data on ~990 cancer cell lines revealed enhanced sensitivity of insulin-like growth factor 1 receptor/ Insulin Receptor (IGF-1R/IR) tyrosine kinase inhibitors (TKIs) in colon cancer cells. Interestingly, ß-catenin/TCF(T cell factor)-responsive promoter activity exhibited a significant positive association with IGF-1R/IR TKI response, while the mutational status of direct upstream genes, such as CTNNB1 and APC, was not significantly associated with the response. The ß-catenin/TCF activity high cell lines express components of IGF-1R/IR signaling more than the low cell lines explaining their enhanced sensitivity against IGF-1R/IR TKI. Reinforcing ß-catenin/TCF responsive promoter activity by introducing CTNNB1 gain-of-function mutations into IGF-1R/IR TKI-resistant cells increased the expression and activity of IGF-1R/IR signaling components and also sensitized the cells to IGF-1R/IR TKIs in vitro and in vivo. Analysis of TCGA data revealed that the stronger ß-catenin/TCF responsive promoter activity was associated with higher IGF-1R and IGF2 transcription in human colon cancer specimens as well. Collectively, compared to the mutational status of upstream genes, ß-catenin/TCF responsive promoter activity has potential to be a stronger predictive positive biomarker for IGF-1R/IR TKI responses in colon cancer cells. The present study highlights the potential of transcriptional activity as therapeutic biomarkers for targeted therapies, overcoming the limited ability of upstream genetic mutations to predict responses.

ATM/CHK/p53 Pathway Dependent Chemopreventive and Therapeutic Activity on Lung Cancer by Pterostilbene.

Among the many stilbenoids found in a variety of berries, resveratrol and pterostilbene are of particular interest given their potential for use in cancer therapeutics and prevention. We purified four stilbenoids from R. undulatum and found that pterostilbene inhibits cancer cell proliferation more efficiently than rhapontigenin, piceatannol and resveratrol. To investigate the underlying mechanism of this superior action of pterostilbene on cancer cells, we utilized a reverse-phase protein array followed by bioinformatic analysis and found that the ATM/CHK pathway is modified by pterostilbene in a lung cancer cell line. Given that ATM/CHK signaling requires p53 for its biological effects, we hypothesized that p53 is required for the anticancer effect of pterostilbene. To test this hypothesis, we used two molecularly defined precancerous human bronchial epithelial cell lines, HBECR and HBECR/p53i, with normal p53 and suppressed p53 expression, respectively, to represent premalignant states of squamous lung carcinogenesis. Pterostilbene inhibited the cell cycle more efficiently in HBECR cells compared to HBECR/p53i cells, suggesting that the presence of p53 is required for the action of pterostilbene. Pterostilbene also activated ATM and CHK1/2, which are upstream of p53, in both cell lines, though pterostilbene-induced senescence was dependent on the presence of p53. Finally, pterostilbene more effectively inhibited p53-dependent cell proliferation compared to the other three stilbenoids. These results strongly support the potential chemopreventive effect of pterostilbene on p53-positive cells during early carcinogenesis.

The Relationship between Prevalence of Osteoporosis and Proportion of Daily Protein Intake.

BACKGROUND: The association between daily protein intake and osteoporosis is still controversial and only a few studies have explored the issue in Korea. This study investigated the relationship between daily protein intake and the prevalence of osteoporosis in Korean adults. METHODS: This study analyzed data extracted from the Korean National Health and Nutrition Examination Survey 4. Participants were aged 19 years or older and had never been treated for osteoporosis. The percentage of calories coming from protein intake was assessed by 24-hour recall method, and participants were divided into three groups according to recommended daily dietary protein intake as a proportion of total daily calories (i.e., <10%, 10%-20%, and >20%). A lumbar or femur neck bone mineral density T-score less than -2.5 was indicative of the presence osteoporosis. The influence of daily protein intake on the prevalence of osteoporosis was analyzed. RESULTS: IN BOTH SEXES, THE GROUP WITH THE HIGHEST PROTEIN INTAKE HAD SIGNIFICANTLY LOWER ODDS OF DEVELOPING LUMBER OSTEOPOROSIS WHEN COMPARED TO THE GROUP WITH THE LOWEST PROTEIN INTAKE, AFTER ADJUSTING FOR ASSOCIATED FACTORS (FEMALES: odds ratio [OR], 0.618; 95% confidence interval [CI], 0.610 to 0.626; P for trend <0.001; males: OR, 0.695; 95% CI, 0.685 to 0.705; P for trend <0.001). CONCLUSION: Sufficient daily protein intake lowered the prevalence of osteoporosis in Korean adults. Further prospective studies are necessary to verify the preventive effect of adequate protein intake on osteoporosis.