Treatment of Ras-induced cancers by the F-actin cappers tensin and chaetoglobosin K, in combination with the caspase-1 inhibitor N1445.
For transforming normal fibroblasts to malignant cells, oncogenic Ras mutants such as v-Ha-ras require Rho family GTPases (Rho, Rac, and CDC42) that are responsible for controlling actin-cytoskeleton organization. Ras activates Rac through a PI-3 kinase-mediated pathway. Rac causes uncapping of actin filaments (F-actin) at the plus-ends, through phosphatidylinositol 4,5 bisphosphate (PIP2), and eventually induces membrane ruffling. Several distinct F-actin/PIP2-binding proteins, such as gelsolin, which severs and caps the plus-ends of actin filaments, or HS1, which cross-links actin filaments, have been shown to suppress v-Ha-Ras-induced malignant transformation when they are overexpressed. Interestingly, an F-actin cross-linking drug (photosensitizer) called MKT-077 suppresses Ras transformation. Thus, an F-actin capping/severing drug might also have an anticancer potential.


OBJECTIVE
This study was conducted to determine first whether Ras-induced malignant phenotype (anchorage-independent growth) is suppressed by overexpression of the gene encoding a large plus-end F-actin capping protein called tensin and second to test the anti-Ras potential of a unique fungal antibiotic (small compound) called chaetoglobosin K (CK) that also caps the plus-ends of actin filaments.


RESULTS
DNA transfection with a retroviral vector carrying the tensin cDNA was used to overexpress tensin in v-Ha-Ras-transformed NIH 3T3 cells. All stable tensin transfectants rarely formed colonies in soft agar, indicating that tensin suppresses the anchorage-independent growth. The anti-Ras action of CK was determined by incubating the Ras-transformants in the presence of CK in soft agar. Two microM CK almost completely inhibited their colony formation, indicating that CK also suppresses the malignant phenotype. However, unlike tensin, CK causes an apoptosis of Ras-transformed NIH 3T3 cells and, less effectively, of normal NIH 3T3 cells, indicating that CK has an F-actin capping-independent side effect(s). CK-induced apoptosis is at least in part caused by CK-induced inhibition of the kinase PKB/AKT. However, a specific ICE/caspase-1 inhibitor called N1445 completely abolished the CK-induced apoptosis by reactivating PKB, but without affecting the CK-induced suppression of Ras transformation.


CONCLUSIONS
Like the F-actin cross-linking drug MKT-077, the F-actin capping drug CK may be useful for the treatment of Ras-associated cancers if it is combined with the ICE inhibitor N1445, which abolishes the side effect of CK. Our observations that two distinct F-actin capping molecules (i.e., tensin and CK) suppress Ras-induced malignant phenotype strongly suggest, if not prove, that capping of actin filaments at the plus-ends alone is sufficient to block one of the Ras signaling pathways essential for its oncogenicity. This notion is compatible with the fact that Ras induces the uncapping of actin filaments at the plus-ends through the Rac/PIP2 pathway.
Tikoo
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A
Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Australia.
Cutler
H
H
Lo
S H
SH
Chen
L B
LB
Maruta
H
H
D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis; D061945:Caspase Inhibitors; D002471:Cell Transformation, Neoplastic / Q000139:chemically induced; D003572:Cytochalasins / Q000494:pharmacology*; D018076:DNA, Complementary / Q000378:metabolism; D011905:Genes, ras / Q000235:genetics*; D005947:Glucose / Q000493:pharmacokinetics; D026121:Indole Alkaloids; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism / Q000494:pharmacology*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000070876:Tensins; D013997:Time Factors; D014162:Transfection; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*
D061945:Caspase Inhibitors; D003572:Cytochalasins; D018076:DNA, Complementary; D026121:Indole Alkaloids; D007211:Indoles; D008840:Microfilament Proteins; C402747:N1445; D019269:Phosphatidylinositol 4,5-Diphosphate; D011518:Proto-Oncogene Proteins; D000070876:Tensins; C019290:chaetoglobosins; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020744:rac GTP-Binding Proteins; D018631:ras Proteins; D005947:Glucose