Hepatocellular carcinoma represents the third common cause of cancer death and the
incidence is steadily increasing. Standard treatments are limited due to frequent
tumor recurrence and a poor responsiveness to conventional chemotherapy. The approval
of sorafenib, a small multikinase inhibitor as the first active systemic treatment
against HCC, increases survival of HCC patients by less than 3 months. Thus, there
is a strong need to understand the molecular mechanisms of sorafenib sensitivity and
resistance in order to instruct sorafenib basedcombination therapies with higher therapeutic
efficacy.
Activation of the Ras/MAPK signaling pathway was reported to play a major role in
liver tumor development and progression. We found that sorafenib showed moderate but
distinct treatment responses in a murine HCC mouse model (NrasG12V driven, p19Arf
deficient HCCs), resembling the response rates of sorafenib treated human HCCs.
To identify genes mediating resistance or sensitivity towards sorafenib, an in vivo
RNAi screen was conducted. Pools of shRNAs targeting genes found amplified in human
hepatocellular carcinomas were introduced into p19Arf-/- mice harboring NrasG12V expressing
liver carcinomas with stable expression of shRNA library pools were either treated
with sorafenib or carrier. After 5 weeks of treatment, shRNA distribution was quantified
in tumors from both cohorts using deep sequencing, whereas depleted shRNAs pinpoint
potential resistance genes towards sorafenib treatment.
Validation experiments with single shRNAs found depleted in sorafenib treated mice
showed
a significant survival advantage compared to non-targeting control shRNA under sorafenib
treatment. Our top-scoring candidate gene represents a kinase (Mapk14) which allowed
us to perform a combination therapy with commonly available inhibitors that results
in a significant survival advantage.
The identified new combination treatment was also expanded to murine HCC cell lines
with additional genetic backgrounds. In these cell lines, therapeutic efficacy as
a result of sorafenib and Mapk14 blockade was also found.Additionally, it was shown
that the newly identified combination therapy significantly decreased proliferation
and induced cell death in a panel of well established human hepatoma cell lines.
To characterize how Mapk14 blockade sensitizes towards sorafenib treatment, microarray
based mRNA expression analyses were conducted and analyzed using Ingenuity pathway
analysis software. This analysis along with genetic validation experiments identified
the Mapk14 downstream target and transcription factor Atf2 as relevant for Mapk14
mediated resistance towards sorafenib treatment.
In summary, our study establishes a new sorafenib based combination therapy for the
treatment of hepatocellular carcinoma and highlights the utility of RNAi loss-of-function
screening for revealing new therapeutic agents in cancer therapy.
