Wnt/ß-catenin Signaling Inhibitors suppress the Tumor-initiating properties of a CD44+CD133+ subpopulation of Caco-2 cells.

Tumor-initiating cells or cancer stem cells are a subset of cancer cells that have tumorigenic potential in human cancer. Although several markers have been proposed to distinguish tumor-initiating cells from colorectal cancer cells, little is known about how this subpopulation contributes to tumorigenesis. Here, we characterized a tumor-initiating cell subpopulation from Caco-2 colorectal cancer cells. Based on the findings that Caco-2 cell subpopulations express different cell surface markers, we were able to discriminate three main fractions, CD44-CD133-, CD44-CD133+, and CD44+CD133+ subsets, and characterized their biochemical and tumorigenic properties. Our results show that CD44+CD133+ cells possessed an unusual capacity to proliferate and could form tumors when transplanted into NSG mice. Additionally, primary tumors grown from CD44+CD133+ Caco-2 cells contained mixed populations of CD44+CD133+ and non-CD44+CD133+ Caco-2 cells, indicating that the full phenotypic heterogeneity of the parental Caco-2 cells was re-created. Notably, only the CD44+CD133+ subset of Caco-2-derived primary tumors had tumorigenic potential in NSG mice, and the tumor growth of CD44+CD133+ cells was faster in secondary xenografts than in primary transplants. Gene expression analysis revealed that the Wnt/ß-catenin pathway was over-activated in CD44+CD133+ cells, and the growth and tumorigenic potential of this subpopulation were significantly suppressed by small-molecule Wnt/ß-catenin signaling inhibitors. Our findings suggest that the CD44+CD133+ subpopulation from Caco-2 cells was highly enriched in tumorigenic cells and will be useful for investigating the mechanisms leading to human colorectal cancer development.

Prognostic impact of EGFR mutation in non-small-cell lung cancer patients with family history of lung cancer.

BACKGROUND: A family history can be a valuable tool in the era of precision medicine. Although a few studies have described an association of family history of lung cancer with EGFR activating mutation, their impact on survival of lung cancer patients is unclear. METHODS: The study included consecutive 829 non-small-cell lung cancer patients who received analysis of EGFR mutation in a prospective lung cancer cohort. Family history of lung cancer was obtained by face-to-face interviews at the time of diagnosis. An association of EGFR activating mutation with a family history of lung cancer in first-degree relatives was evaluated with multivariate logistic regression analysis, and its association with survival was estimated with Cox's proportional hazards model. RESULTS: Seventy five (9.0%) patients had family history of lung cancer. The EGFR mutation was commonly observed in patients with positive family history compared to those with no family history (46.7% v 31.3%, χ2 p = 0.007). The family history was significantly associated with the EGFR mutation (aOR and 95% CI: 2.01 and 1.18-3.60, p = 0.011). Patients with the positive family history survived longer compared to those without (MST, 17.9 v 13.0 months, log-rank p = 0.037). The presence of the EGFR mutation was associated with better survival in patients without the family history (aHR and 95% CI: 0.72 and 0.57-0.90, p = 0.005). However, this prognostic impact was not observed in patients with the positive family history (aHR and 95% CI: 1.01 and 0.50-2.36, p = 0.832). CONCLUSIONS: In comparison to patients without the family history, EGFR activating mutation was common, and it did not affect prognosis in patients with positive family history.

The reprogramming factor nuclear receptor subfamily 5, group A, member 2 cannot replace octamer-binding transcription factor 4 function in the self-renewal of embryonic stem cells.

Although octamer-binding transcription factor 4 (Oct-4) is one of the most intensively studied factors in mammalian development, no cellular genes capable of replacing Oct-4 function in embryonic stem (ES) cells have been found. Recent data show that nuclear receptor subfamily 5, group A, member 2 (Nr5a2) is able to replace Oct-4 function in the reprogramming process; however, it is unclear whether Nr5a2 can replace Oct-4 function in ES cells. In this study, the ability of Nr5a2 to maintain self-renewal and pluripotency in ES cells was investigated. Nr5a2 localized to the nucleus in ES cells, similarly to Oct-4. However, expression of Nr5a2 failed to rescue the stem cell phenotype or to maintain the self-renewal ability of ES cells. Furthermore, as compared with Oct-4-expressing ES cells, Nr5a2-expressing ES cells showed a reduced number of cells in S-phase, did not expand normally, and did not remain in an undifferentiated state. Ectopic expression of Nr5a2 in ES cells was not able to activate transcription of ES cell-specific genes, and gene expression profiling demonstrated differences between Nr5a2-expressing and Oct-4-expressing ES cells. In addition, Nr5a2-expressing ES cells were not able to form teratomas in nude mice. Taken together, these results strongly suggest that the gene regulation properties of Nr5a2 and Oct-4 and their abilities to confer self-renewal and pluripotency of ES cells differ. The present study provides strong evidence that Nr5a2 cannot replace Oct-4 function in ES cells.

Autophagy regulates homeostasis of pluripotency-associated proteins in hESCs.

The pluripotency of embryonic stem cells (ESCs) is maintained by intracellular networks of many pluripotency-associated (PA) proteins such as OCT4, SOX2, and NANOG. However, the mechanisms underlying the regulation of protein homeostasis for pluripotency remain elusive. Here, we first demonstrate that autophagy acts together with the ubiquitin-proteasome system (UPS) to modulate the levels of PA proteins in human ESCs (hESCs). Autophagy inhibition impaired the pluripotency despite increment of PA proteins in hESCs. Immunogold-electron microscopy confirmed localization of OCT4 molecules within autophagosomes. Also, knockdown of LC3 expression led to accumulation of PA proteins and reduction of pluripotency in hESCs. Interestingly, autophagy and the UPS showed differential kinetics in the degradation of PA proteins. Autophagy inhibition caused enhanced accumulation of both cytoplasmic and nuclear PA proteins, whereas the UPS inhibition led to preferentially degrade nuclear PA proteins. Our findings suggest that autophagy modulates homeostasis of PA proteins, providing a new insight in the regulation of pluripotency in hESCs.