Functional analysis and therapeutic targeting of AKT isoforms in BRAF mutant melanoma
recent advances, metastatic melanoma remains the deadliest form of skin cancer, and new
therapeutic strategies are urgently needed. The PI3K/AKT pathway is known to promote
tumor progression and metastatic dissemination in many cancer types, including melanoma.
PI3K/AKT signaling is hyperactivated in many melanomas, often through loss of the
negative regulator PTEN, which ... read moreoccurs in 12-20% of cases. PTEN loss cooperates with
oncogenic BRAF to induce metastatic melanoma and leads to unrestrained activity of the
PI3K effector kinase, AKT. The AKT family of serine/threonine kinases comprises three
highly homologous isoforms (AKT1, AKT2, and AKT3) that are major effectors of the PI3K
pathway, but despite their unique roles in other cancers, isoform-specific effects in
melanoma have yet to be systematically interrogated. We performed shRNA mediated
conditional knockdown and CRISPR/Cas9 gene editing of AKTs in a wide variety of human
melanoma cell lines, as well as genetic ablation of each isoform in a BRAF-driven mouse
model of melanoma. We reveal a role for AKT2 in promoting melanoma cell migration and
invasion in vitro and find that AKT2 is required for metastatic seeding in vivo.
Additionally, AKT2 specific depletion delays tumor growth and improves survival of mice
with metastatic disease after tumor cell seeding, suggesting that AKT2 supports growth
or survival in the metastatic niche. We propose several mechanisms whereby AKT2 may
mediate these phenotypes, including via regulation of key epithelial-mesenchymal genes,
promoting aerobic glycolysis, and responding to hypoxia. In contrast, we observe that
the AKT1 isoform specifically promotes melanoma cell proliferation, and genetic ablation
of AKT1 in melanoma prone mice prolongs overall survival. We also reveal that the AKT3
isoform may be important in UV-initiated melanomagenesis. Lastly, while non-specific
pan-AKT inhibitors are used clinically to moderate benefit, their use is hampered by
myriad off-target effects. To increase the efficacy of AKT targeting for clinical
benefit, we endeavored to identify AKT isoform-specific substrate effectors that may be
mediating differential phenotypes, and therefore potential targets for therapy. We also
report efforts to use a tumor specific antigen to target a clinically relevant pan-AKT
inhibitor to melanoma tumors in the mouse, and additionally test small molecule
inhibitors that synergize with existing targeted therapies. In summary, our work moves
toward establishing the contribution of AKT isoforms to melanoma, to improve therapeutic
strategies and outcome for this devastating
Thesis (Ph.D.)--Tufts University, 2018.
Submitted to the Dept. of Genetics.
Advisors: Phil Hinds, and Karl Munger.
Committee: Philip Tsichlis, Charlotte Kuperwasser, and Alex Toker.
Keywords: Genetics, Oncology, and Cellular biology.read less