bafilomycin-a and Neoplasm-Metastasis

KRAS mutations contribute to cell proliferation and survival in numerous cancers, including colorectal cancers (CRC). One pathway through which mutant KRAS acts is an inflammatory pathway that involves the kinase IKK and activates the transcription factor NF-κB. BRAF, a kinase that is downstream of KRAS, is mutated in a subset of CRC and is predictive of poor prognosis and therapeutic resistance. We found that, in contrast to mutant KRAS, mutant BRAF (BRAF(V600E)) did not trigger NF-κB activation but instead triggered the phosphorylation of a proteolytic fragment of IKKα (p45-IKKα) in CRC cells. BRAF(V600E) CRC cells had a high abundance of phosphorylated p45-IKKα, which was decreased by a RAF inhibitor. However, the abundance and DNA binding of NF-κB in these cells were unaffected by the RAF inhibitor, and expression of BRAF(V600E) in human embryonic kidney-293T cells did not activate an NF-κB reporter. Moreover, BRAF-induced transformation of NIH-3T3 cells and BRAF-dependent transcription required phosphorylation of p45-IKKα. The kinase TAK1, which was associated with the endosomal compartment, phosphorylated p45-IKKα. Inhibition of endosomal vacuolar adenosine triphosphatase (V-ATPase) with chloroquine or bafilomycin A1 blocked p45-IKKα phosphorylation and induced apoptosis in BRAF-mutant CRC cells independent of autophagy. Treating mice with V-ATPase inhibitors reduced the growth and metastasis of BRAF(V600E) xenograft tumors in the cecum of mice.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Chloroquine; DNA; Dose-Response Relationship, Drug; Endosomes; HEK293 Cells; Humans; I-kappa B Kinase; Inflammation; Macrolides; Mice; Mice, Knockout; Mutation; Neoplasm Metastasis; Neoplasm Transplantation; NF-kappa B p50 Subunit; NIH 3T3 Cells; Phosphorylation; Prognosis; Proto-Oncogene Proteins B-raf

We investigated whether alterations in the mechanisms involved in intracellular pH (pHin) and intracellular calcium ([Ca2+]in) homeostasis are associated with the metastatic potential of poorly (A375P) and highly (C8161) metastatic human melanoma cells. We monitored pHin and [Ca2+]in simultaneously, using the fluorescence of SNARF-1 and Fura-2, respectively. Our results indicated that steady-state pHin and [Ca2+]in between these cell types were not significantly different. Treatment of cells with NH4Cl resulted in larger pHin increases in highly than in poorly metastatic cells, suggesting that C8161 cells have a lower H+ buffering capacity than A375P. NH4Cl treatment also increased [Ca2+]in only in C8161 cells. To determine if the changes in [Ca2+]in triggered by NH4Cl treatment were due to alterations in either H+- or Ca2+-buffering capacity, cells were treated with the Ca2+-ionophore 4Br-A23187, to alter [Ca2+]in. The magnitude of the ionophore-induced [Ca2+]in increase was slightly greater in C8161 cells than in A375P. Moreover, A375P cells recover from the ionophore-induced [Ca2+]in load, whereas C8161 cells did not, suggesting that A375P may exhibit distinct [Ca2+]in regulatory mechanisms than C8161 cells, to recover from Ca2+ loads. Removal of extracellular Ca2+ ([Ca2+]ex) decreased [Ca2+]in in both cell types at the same extent. Ionophore treatment in the absence of [Ca2+]ex transiently increased [Ca2+]in in C8161, but not in A375P cells. Endoplasmic reticulum (ER) Ca2+-ATPase inhibitors such as cyclopiazonic acid (CPA) and thapsigargin (TG) increased steady-state [Ca2+]in only in C8161 cells. Together, these data suggest that the contribution of intracellular Ca2+ stores for [Ca2+]in homeostasis is greater in highly than in poorly metastatic cells. Bafilomycin treatment, to inhibit V-type H+-ATPases, corroborated our previous results that V-H+-ATPases are functionally expressed at the plasma membranes of highly metastatic, but not in poorly metastatic cells (Martínez-Zaguilán et al., 1993). Collectively, these data suggest that distinct pHin and [Ca2+]in regulatory mechanisms are present in poorly and highly metastatic human melanoma cells.

Topics: Ammonium Chloride; Anti-Bacterial Agents; Benzopyrans; Calcimycin; Calcium; Calcium-Transporting ATPases; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Homeostasis; Humans; Hydrogen-Ion Concentration; Indoles; Ionophores; Macrolides; Melanoma; Naphthols; Neoplasm Metastasis; Proton-Translocating ATPases; Rhodamines; Thapsigargin; Tumor Cells, Cultured