Chemical genetic blockade of transformation reveals dependence on aberrant oncogenic signaling.
BACKGROUND
Our understanding of protein kinase inhibition in the treatment of cancer is clearly limited by the lack of inhibitors that selectively block a single kinase implicated in neoplastic transformation. One approach to developing specific inhibitors is to engineer in protein kinases silent mutations that allow selective inhibition while retaining kinase activity. Because it is implicated in a large number of malignancies, EGFR provides an attractive target for such selective kinase inhibition.


RESULTS
We generated an inhibitor-sensitized allele of the transforming receptor tyrosine kinase v-erbB. Transformation of immortalized rodent fibroblasts by sensitized versions of v-erbB (v-erbB-as1) was blocked by 1-napthyl PP1 (NaPP1), a cell-permeable ATP-competitive inhibitor. NaPP1 also reversed morphological transformation by v-erbB-as1. Signaling through MAP kinase and PI(3) kinase was initially blocked by inhibitor treatment and then recovered to levels comparable to those in nontransformed cells. Surprisingly, NaPP1-treated v-erbB-as1 cells failed to re-enter the cell cycle, showed decreased levels of D- and A-type cyclins, and showed increased levels of p27. To extend this result, we showed that NaPP1 treatment of v-Src-as1 cells also led to cell cycle arrest. Arrested cells could be rescued with a conditional allele of Raf or by transduction of a constitutive allele of cyclin D1.


CONCLUSIONS
These data suggest that mammalian cells can become dependent on aberrant oncogenic signaling; this dependency renders them incapable of returning to a normal, proliferative phenotype.
Department of Neurology, University of California, San Francisco, CA 94143, USA.
Qi-Wen
QW
Fan
Chao
C
Zhang
Kevan M
KM
Shokat
William A
WA
Weiss
D016475:3T3 Cells; D000483:Alleles; D000818:Animals; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism; D002455:Cell Division / Q000502:physiology; D048429:Cell Size; D002471:Cell Transformation, Neoplastic; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D018773:Genes, erbB-1 / Q000235:genetics*; D007150:Immunohistochemistry; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009281:Naphthalenes / Q000378:metabolism; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics / Q000378:metabolism; D018739:Oncogene Proteins v-erbB / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D025521:Tumor Suppressor Proteins / Q000378:metabolism
C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D016213:Cyclins; D004791:Enzyme Inhibitors; D009281:Naphthalenes; D018739:Oncogene Proteins v-erbB; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D015688:Oncogene Protein pp60(v-src); D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases