Anti-EGFR antibody efficiently and specifically inhibits human TSC2-/- smooth muscle cell proliferation. Possible treatment options for TSC and LAM.
BACKGROUND
Tuberous sclerosis complex (TSC), a tumor syndrome caused by mutations in TSC1 or TSC2 genes, is characterized by the development of hamartomas. We previously isolated, from an angiomyolipoma of a TSC2 patient, a homogenous population of smooth muscle-like cells (TSC2(-/-) ASM cells) that have a mutation in the TSC2 gene as well as TSC2 loss of heterozygosity (LOH) and consequently, do not produce the TSC2 gene product, tuberin. TSC2(-/-) ASM cell proliferation is EGF-dependent.


RESULTS
Effects of EGF on proliferation of TSC2(-/-) ASM cells and TSC2(-/-) ASM cells transfected with TSC2 gene were determined. In contrast to TSC2(-/-) ASM cells, growth of TSC2-transfected cells was not dependent on EGF. Moreover, phosphorylation of Akt, PTEN, Erk and S6 was significantly decreased. EGF is a proliferative factor of TSC2(-/-) ASM cells. Exposure of TSC2(-/-) ASM cells to anti-EGFR antibodies significantly inhibited their proliferation, reverted reactivity to HMB45 antibody, a marker of TSC2(-/-) cell phenotype, and inhibited constitutive phosphorylation of S6 and ERK. Exposure of TSC2(-/-) ASM cells to rapamycin reduced the proliferation rate, but only when added at plating time. Although rapamycin efficiently inhibited S6 phosphorylation, it was less efficient than anti-EGFR antibody in reverting HMB45 reactivity and blocking ERK phosphorylation. In TSC2(-/-) ASM cells specific PI3K inhibitors (e.g. LY294002, wortmannin) and Akt1 siRNA had little effect on S6 and ERK phosphorylation. Following TSC2-gene transfection, Akt inhibitor sensitivity was observed.


CONCLUSIONS
Our results show that an EGF independent pathway is more important than that involving IGF-I for growth and survival of TSC(-/-) ASM cells, and such EGF-dependency is the result of the lack of tuberin.
Lesma
Elena
E
Laboratory of Pharmacology , Department of Medicine, Surgery and Dentistry- Polo H. San Paolo, Faculty of Medicine, University of Milan, Milan, Italy. elena.lesma@unimi.it
Grande
Vera
V
Ancona
Silvia
S
Carelli
Stephana
S
Di Giulio
Anna Maria
AM
Gorio
Alfredo
A
D000328:Adult; D000906:Antibodies / Q000494:pharmacology* / Q000627:therapeutic use; D000915:Antibody Affinity; D000918:Antibody Specificity; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D066246:ErbB Receptors / Q000276:immunology*; D005260:Female; D006801:Humans; D018192:Lymphangioleiomyomatosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D008954:Models, Biological; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D018235:Smooth Muscle Tumor / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D014162:Transfection; D014402:Tuberous Sclerosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
D000906:Antibodies; C000624653:TSC2 protein, human; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; C512478:EGFR protein, human; D066246:ErbB Receptors