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Supplementary data of in vitro cell line experiments, human stomach tumor database/sample analysis and animal experiments.

Supplementary Table 1: Antibodies and proteins used in the present study

Supplementary Fig 6 related to Fig 6: PCa tissues with elevated WNT signaling activation express higher level of hTERT

Supplementary Mehods, Materials, figures and tables Figure S1. Immunofluorescent staining of Numb in PCa tissues. Figure S2. Classical Wnt signaling was not affected by Numb in PCa cells. Figure S3. The mRNA levels of Hes1 and GLI1 negatively correlated with Numb in PCa tissues. Figure S4. Identification of Numb proximal promoter. Figure S5. Notch signaling exerted no significant effect on the transcription of Numb gene. Figure S6. Effects of the treatment of DAPT, cyclopamine, or a combination of DAPT and cyclopamine on the GFP status of Numb-promoter-GFP reporter lentivirus transfected C4-2B cells. Figure S7. Treatment with a combination of DAPT and cyclopamine resulted in a shift of Numb-/low to Numbhigh cells. Figure S8. The percentage of cells with nuclear localized AR protein was significantly lower in Numb-/low PCa cells than Numbhigh cells. Figure S9. Blockage of Notch and Hedgehog signaling by DAPT and cyclopamine significantly enhanced the apoptosis of Numb-/low cells induced by flutamide-mediated androgen deprivation. Supplementary Table 1. Detailed clinical information of human prostate cancer patients in this study Supplementary Table 2. Primer sequences for qRT-PCR Supplementary Table 3. Antibodies used in Apoptosis Assay, Immunoblotting and Immunofluorescence experiments. Supplementary Table 4. shPTCH1 and shNumb sequences. Supplementary Table 5. Information of plasmids components of RBP-Jκ, GLI and TCF/LEF1 luciferase reporter Supplementary Table 6. Information of response elements of RBP-Jκ, GLI and TCF/LEF1 luciferase reporter

Supplementary Table 2: Primer sequences used in the qRT-PCR experiment

Supplementary Fig 7 related to Fig 7: Validation of beta-catenin knockdown efficiency in PCa cells

Supplementary Fig 4 related to Fig 4: hTERThigh PCa cells exhibit three distinct cell division modes in a Numb-dsRed segregation context

Cancer stem-like cells (CSC) drive cancer progression and recurrence. Self-renewal expansion of CSC is achieved through symmetric cell division, yet how external stimuli affect intracellular regulatory programs of CSC division modes and stemness remains obscure. Here, we report that the hTERThigh prostate cancer cells exhibit CSC properties, including a stem cell–associated gene expression signature, long-term tumor-propagating capacity and epithelial-to-mesenchymal transition. In promoting the self-renewal symmetric division of hTERThigh prostate cancer cells, WNT3a dramatically decreased the ratio of hTERThigh prostate cancer cells undergoing asymmetric division. Increased WNT/β-catenin signal activation was also detected in hTERThigh prostate cancer cells. hTERT-mediated CSC properties were at least partially dependent on β-catenin. These findings provide novel cellular and molecular mechanisms for the self-renewal of CSC orchestrated by tumor microenvironmental stimuli and intracellular signals. Cancer Res; 77(9); 2534–47. ©2017 AACR.

hTERT-/low and hTERThigh PCa cell division modes from WNT/β-Catenin Directs Self-Renewal Symmetric Cell Division of hTERThigh Prostate Cancer Stem Cells

Supplementary Fig 5 related to Fig 5: The Effects of WNT ligands and WNT inhibitors on SCD I, SCD II and ACD ratio of hTERThigh LNCaP cells

Dysregulation of Wnt/β-catenin signaling is frequently observed in human gastric cancer. Elucidation of the tumor immune microenvironment is essential for understanding tumorigenesis and for the development of immunotherapeutic strategies. However, it remains unclear how β-catenin signaling regulates the tumor immune microenvironment in the stomach. Here, we identify CCL28 as a direct transcriptional target gene of β-catenin/T-cell factor (TCF). Protein levels of β-catenin and CCL28 positively correlated in human gastric adenocarcinoma. β-Catenin–activated CCL28 recruited regulatory T (Treg) cells in a transwell migration assay. In a clinically relevant mouse gastric cancer model established by Helicobacter (H.) felis infection and N-methyl-N-nitrosourea (MNU) treatment, inhibition of β-catenin/TCF activity by a pharmacologic inhibitor iCRT14 suppressed CCL28 expression and Treg cell infiltration in the stomach. Moreover, an anti-CCL28 antibody attenuated Treg cell infiltration and tumor progression in H. felis/MNU mouse models. Diphtheria toxin–induced Treg cell ablation restrained gastric cancer progression in H. felis/MNU-treated DEREG (Foxp3-DTR) mice, clarifying the tumor-promoting role of Treg cells. Thus, the β-catenin–CCL28–Treg cell axis may serve as an important mechanism for immunosuppression of the stomach tumor microenvironment. Our findings reveal an immunoregulatory role of β-catenin signaling in stomach tumors and highlight the therapeutic potential of CCL28 blockade for the treatment of gastric cancer.Significance:These findings demonstrate an immunosuppressive role of tumor-intrinsic β-catenin signaling and the therapeutic potential of CCL28 blockade in gastric cancer.

Supplementary Materials and Methods

Supplementary Fig 2 related to Fig 2: hTERT promotes xenograft tumor growth and EMT of PCa cells

Supplementary Fig 8 working model: Schematic model of hTERThigh PCSCs cell division modes modulated by WNT/beta-catenin

Supplementary Fig 1 related to Fig 1: hTERT expression in PCa specimens and FACS sorted hTERT-/low and hTERThigh PCa cells

Supplementary Fig 3 related to Fig 3: hTERT enhances the holoclone formation capacity of PCa cells

Dysregulation of Wnt/β-catenin signaling is frequently observed in human gastric cancer. Elucidation of the tumor immune microenvironment is essential for understanding tumorigenesis and for the development of immunotherapeutic strategies. However, it remains unclear how β-catenin signaling regulates the tumor immune microenvironment in the stomach. Here, we identify CCL28 as a direct transcriptional target gene of β-catenin/T-cell factor (TCF). Protein levels of β-catenin and CCL28 positively correlated in human gastric adenocarcinoma. β-Catenin–activated CCL28 recruited regulatory T (Treg) cells in a transwell migration assay. In a clinically relevant mouse gastric cancer model established by Helicobacter (H.) felis infection and N-methyl-N-nitrosourea (MNU) treatment, inhibition of β-catenin/TCF activity by a pharmacologic inhibitor iCRT14 suppressed CCL28 expression and Treg cell infiltration in the stomach. Moreover, an anti-CCL28 antibody attenuated Treg cell infiltration and tumor progression in H. felis/MNU mouse models. Diphtheria toxin–induced Treg cell ablation restrained gastric cancer progression in H. felis/MNU-treated DEREG (Foxp3-DTR) mice, clarifying the tumor-promoting role of Treg cells. Thus, the β-catenin–CCL28–Treg cell axis may serve as an important mechanism for immunosuppression of the stomach tumor microenvironment. Our findings reveal an immunoregulatory role of β-catenin signaling in stomach tumors and highlight the therapeutic potential of CCL28 blockade for the treatment of gastric cancer. Significance: These findings demonstrate an immunosuppressive role of tumor-intrinsic β-catenin signaling and the therapeutic potential of CCL28 blockade in gastric cancer.

Purpose: To elucidate the role and molecular mechanism of Numb in prostate cancer and the functional contribution of Numb−/low prostate cancer cells in castration resistance. Experimental Design: The expression of Numb was assessed using multiple Oncomine datasets and prostate cancer tissues from both humans and mice. The biological effects of the overexpression and knockdown of Numb in human prostate cancer cell lines were investigated in vitro and in vivo. In addition, we developed a reliable approach to distinguish between prostate cancer cell populations with a high or low endogenous expression of Numb protein using a Numb promoter–based lentiviral reporter system. The difference between Numb−/low and Numbhigh prostate cancer cells in the response to androgen-deprivation therapy (ADT) was then tested. The likely downstream factors of Numb were analyzed using luciferase reporter assays, immunoblotting, and quantitative real-time PCR. Results: We show here that Numb was downregulated and negatively correlated with prostate cancer advancement. Functionally, Numb played an inhibitory role in xenograft prostate tumor growth and castration-resistant prostate cancer development by suppressing Notch and Hedgehog signaling. Using a Numb promoter–based lentiviral reporter system, we were able to distinguish Numb−/low prostate cancer cells from Numbhigh cells. Numb−/low prostate cancer cells were smaller and quiescent, preferentially expressed Notch and Hedgehog downstream and stem-cell–associated genes, and associated with a greater resistance to ADT. The inhibition of the Notch and Hedgehog signaling pathways significantly increased apoptosis in Numb−/low cells in response to ADT. Conclusions: Numb−/low enriches a castration-resistant prostate cancer cell subpopulation that is associated with unregulated Notch and Hedgehog signaling. Clin Cancer Res; 23(21); 6744–56. ©2017 AACR.

Cancer stem-like cells (CSC) drive cancer progression and recurrence. Self-renewal expansion of CSC is achieved through symmetric cell division, yet how external stimuli affect intracellular regulatory programs of CSC division modes and stemness remains obscure. Here, we report that the hTERThigh prostate cancer cells exhibit CSC properties, including a stem cell–associated gene expression signature, long-term tumor-propagating capacity and epithelial-to-mesenchymal transition. In promoting the self-renewal symmetric division of hTERThigh prostate cancer cells, WNT3a dramatically decreased the ratio of hTERThigh prostate cancer cells undergoing asymmetric division. Increased WNT/β-catenin signal activation was also detected in hTERThigh prostate cancer cells. hTERT-mediated CSC properties were at least partially dependent on β-catenin. These findings provide novel cellular and molecular mechanisms for the self-renewal of CSC orchestrated by tumor microenvironmental stimuli and intracellular signals. Cancer Res; 77(9); 2534–47. ©2017 AACR.