gdc-0449 and Stomach-Neoplasms

Gastric cancers may harbor a subset of cells with cancer stem cell (CSC) properties, including chemotherapy resistance, and CD44 is a gastric CSC marker. The Hedgehog (HH) pathway is a key developmental pathway that can be subverted by CSCs during tumorigenesis. Here, we examine the role of HH signaling in CD44(+) gastric cancer cells.. Gastric cancer cell lines, tumor xenografts, and patient tumors were examined.. Gastric cancer cell lines AGS, MKN-45, and NCI-N87 grown as spheroids or sorted for CD44(+) were found to have upregulation of HH pathway proteins. HH inhibition using Smoothened (Smo) shRNA or vismodegib (VIS) decreased spheroid formation and colony formation. CD44(+) cells, compared with unselected cells, were also resistant to 5-fluorouracil and cisplatin chemotherapy, and this resistance was reversed in vitro and in xenografts with Smo shRNA or VIS. CD44(+) cells also had significantly more migration, invasion, and anchorage-independent growth, and these properties could all be blocked with HH inhibition. Clinical tumor samples from a phase II trial of chemotherapy with or without VIS for advanced gastric cancer were analyzed for CD44 expression. In the chemotherapy alone group, high CD44 expression was associated with decreased survival, whereas in the chemotherapy plus VIS group, high CD44 expression was associated with improved survival.. HH signaling maintains CSC phenotypes and malignant transformation phenotypes in CD44(+) gastric cancer cells, and HH inhibition can reverse chemotherapy resistance in CD44(+) cells. Gastric cancer is a heterogeneous disease, and the strategy of combining chemotherapy with HH inhibition may only be effective in tumors with high CD44 levels.

Topics: Adenocarcinoma; Anilides; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Cell Movement; Cell Proliferation; Cisplatin; Drug Resistance, Neoplasm; Female; Flow Cytometry; Fluorescent Antibody Technique; Fluorouracil; Hedgehog Proteins; Humans; Hyaluronan Receptors; Leucovorin; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplastic Stem Cells; Prognosis; Pyridines; Receptors, G-Protein-Coupled; RNA, Small Interfering; Signal Transduction; Smoothened Receptor; Spheroids, Cellular; Stomach Neoplasms; Survival Rate; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The sonic hedgehog (Shh) pathway is known to be vital in embryonic development and cancer propagation due to its irreplaceable role in cell proliferation and differentiation. GDC‑0449, a basal cell skin cancer target drug approved by the Food and Drugs Administration, is a smoothened (Smo)-specific antagonist. Although it has been clinically verified as a valid drug for the treatment of skin and pancreatic cancer, the application of GDC‑0449 in gastric cancer requires further investigation. In the present study, high-glucose Dulbecco's modified Eagle's medium with 10% fetal bovine serum was used for routine SGC‑7901 cell line culture. A Cell Counting Kit‑8 assay was employed for determination of the reproductive rate of the cells. Flow cytometry was performed to determine the apoptosis status of the SGC‑7901 cell line through Q4 analysis. Reverse transcription-quantitative polymerase chain reaction and Western blot analyses were used as target molecule detection vehicles. As expected, GDC‑0449 reduced the expression levels of Shh‑associated molecules, including Smo and gli1, compared with the blank group. The rate of cell proliferation was markedly limited and was accompanied by an increase in the apoptotic rate following GDC‑0449 exposure. In addition, further investigations confirmed B cell lymphoma‑2 (Bcl‑2) as the downstream molecular mechanism of GDC‑0449 efficacy. Of note, representatives of the cancer stem cell (CSC) surface marker, CD44 and CD133, demonstrated a similar trend to the Smo restriction observed. By repressing the expression of Bcl‑2, GDC‑0449 inhibited the normal proliferation of SGC‑7901 cells, and accelerated the apoptotic rate of the cells. It may also alter CSC properties due to the reduction in the expression of surface markers.

Topics: Anilides; Apoptosis; Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Neoplastic Stem Cells; Proto-Oncogene Proteins c-bcl-2; Pyridines; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Stomach Neoplasms

Drug resistance in gastric cancer largely results from the gastric cancer stem cells (GCSCs), which could be targeted to improve the efficacy of chemotherapy. In this study, we identified a subpopulation of GCSCs enriched in holoclones that expressed CD44(+)/Musashi-1(+) stem cell biomarkers, capable of self-renewal and proliferation. Enriched CD44(+)/Musashi-1(+) GCSCs demonstrated elevated expression of sonic hedgehog (SHH) and glioma-associated oncogene homolog 1 (GLI1), the well-known signaling pathway molecules involved in the drug resistance. Further, CD44(+)/Musashi-1(+) cells exhibited high drug efflux bump activity and were resistant to doxorubicin (Dox)-induced apoptosis, and unregulated the ATP-binding cassette sub-family G member 2 (ABCG2) expression,. The above effects on apoptosis were reversed in the presence of GLI inhibitors, GANT61 and GDC-0449, or by the knockdown of GLI1/SHH. Upon knockdown of GLI1, expression of ABCG2 was downregulated the antitumor effects were significantly improved as observed in the gastric cancer xenograft. Collectively, our study revealed that co-expression of CD44(+)/Musashi-1(+) could be used to identify GCSCs, which also accounts for the drug resistance in gastric cancer. SHH-GLI and its downstream effector ABCG2 could be better targeted to possibly improve the efficacy of chemotherapy in drug-resistant gastric cancers.

Topics: Anilides; Animals; Antibiotics, Antineoplastic; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cell Line, Tumor; Cell Self Renewal; Doxorubicin; Drug Resistance, Neoplasm; Female; Gene Expression; Hedgehog Proteins; HEK293 Cells; Humans; Hyaluronan Receptors; Mice, Nude; Neoplasm Proteins; Neoplastic Stem Cells; Pyridines; Signal Transduction; Stomach Neoplasms; Transcription Factors; Xenograft Model Antitumor Assays; Zinc Finger Protein GLI1