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.

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.

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.

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.

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.

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.