Examining the Role of Metabolic Pathways as Therapeutic Modalities for Triple Negative Breast Cancer
Author ORCID Identifier
Date Available
7-17-2020
Year of Publication
2020
Degree Name
Doctor of Philosophy (PhD)
Document Type
Doctoral Dissertation
College
Medicine
Department/School/Program
Toxicology and Cancer Biology
First Advisor
Dr. B. Mark Evers
Abstract
Triple negative breast cancer (TNBC) comprises 15-20% of breast cancers, affects a younger patient population than other subtypes, and is very aggressive. TNBC is comprised of a diverse group of tumors that have proven refractory to targeted therapy and can be difficult to treat. Patients generally receive neoadjuvant chemotherapy (NAC), surgery, and radiotherapy. The standard of care for NAC includes a taxane, an anthracycline, and/or cyclophosphamide, and administration of NAC has resulted in pathological complete response (pCR) in 30-40% of patients. However, a majority of TNBC patients will not reach pCR and instead have residual disease (RD), which is associated with worse outcomes. Patients with RD are more likely to experience locoregional or distant recurrence, and they have lower rates of overall survival, breast cancer-specific survival, disease-free survival, and distant relapse-free survival. Five-year survival rates for TNBC are 91% for localized disease, 65% with regional recurrence, and only 11% with distant recurrence. The reductions in survival with metastasis indicate that patients who have RD after receiving NAC desperately need novel interventions. AMP-activated protein kinase (AMPK) and protein kinase B (Akt) are important cellular energy regulators that have been implicated in cancer therapy and progression. However, further work is needed to fully establish their roles as therapeutic modalities in TNBC. This study analyzed whether targeting the AMPK and Akt signaling pathways can enhance TNBC therapy or reduce TNBC metastasis.
Chemical activation of AMPK suppresses growth of cancer cells, but many common AMPK activators—such as AICAR or 2-deoxyglucose—have low sensitivity and require dosing in the millimolar range. This has led to their unsuccessful translation to the clinic. FND-4b is a novel compound that activates AMPK at micromolar concentrations in colorectal cancer cells, but its effects in TNBC are unknown. Treatment of TNBC cells with FND-4b induced AMPK activation and signaling through its downstream pathways. Phosphorylation of acetyl CoA carboxylase (ACC)—a direct target of AMPK—was increased, indicating a decrease in fatty acid synthesis. Furthermore, activation of ribosomal protein S6 was reduced, which suggests reduced flux through the mTOR pathway. FND-4b also suppressed proliferation of TNBC in a dose-dependent manner in the low micromolar range, which is substantially lower than well-known AMPK activators. Finally, FND-4b increased apoptosis induction in TNBC cells at micromolar doses. Taken together, these findings indicate that FND-4b reduces proliferation and induces apoptosis through AMPK activation in TNBC cells at lower doses than many other AMPK activators.
AMPK inhibition has increased sensitivity of cancer cells to radiotherapy due to suppression of autophagy. Chemical inhibition of the PI3K signaling cascade has also potentiated radiation-induced cell death in TNBC cells. However, the ability of individual AMPK or Akt isoforms to sensitize TNBC cells to radiotherapy has not been studied. Moreover, while a double-negative feedback loop exists between AMPK and Akt—a downstream effector of PI3K—the impact of combined inhibition of AMPK and Akt isoforms on TNBC survival after radiation is unknown. Immunohistochemical (IHC) staining indicated that AMPKα1 is expressed in only the cytoplasm of TNBC, while AMPKα2 is found in both the cytoplasm and the nucleus. AMPKα1 or AMPKα2 knockdown decreased proliferation and induced G1 cell cycle arrest in TNBC cells but did not induce apoptosis alone or in combination with radiotherapy. The role of PI3K p85α, p85β, p110α, p110β, Akt1, and Akt2 proteins on TNBC cell cycle progression and apoptosis induction was then analyzed. Akt1 and p110αsuppressed cyclin D1 expression and induced apoptosis. Silencing Akt1 potentiated radiation-induced apoptosis and further suppressed survival of TNBC cells after radiation exposure. Treatment of TNBC cells with the Akt inhibitor MK-2206 48 h after radiotherapy decreased Akt1 expression and promoted synergistic apoptosis induction. Taken together, these results indicate that inhibiting Akt1 expression is a potentially promising approach to enhance TNBC treatment.
Distant spread of TNBC is correlated with a substantial drop in 5-year survival. However, the ability of Akt isoforms to mediate TNBC metastasis is unknown, and the specific steps of metastasis that are regulated by AMPKα isoforms are unclear. In addition, combined inhibition of Akt and AMPKα isoforms to suppress TNBC metastatic spread has not been attempted. IHC staining indicated that Akt1, Akt2, and AMPKα1 were primarily expressed in the cytoplasm of TNBC lymph node metastases. IHC staining also showed that AMPKα2 was mostly expressed in both the cytoplasm and nucleus of TNBC lymph node metastases. Silencing Akt2 reduced expression of the invasive proteins Snail and Claudin-1 in TNBC cells. Moreover, knockdown of Akt1 and Akt2 induced apoptosis in TNBC cells potentially by increasing Bim expression and reducing Mcl-1 expression, respectively. Silencing AMPKα1 or AMPKα2 did not consistently affect the expression of the invasive proteins tested or induce apoptosis in TNBC cells. Suppressing levels of Akt1 or Akt2 significantly reduced TNBC lung colonization independent of Akt activity, but knocking down AMPKα1, AMPKα2, or AMPKα1/2 did not impact the ability of TNBC cells to metastasize to the lungs. Moreover, targeting Akt1 and AMPKα1 together did not decrease TNBC lung metastasis more than silencing Akt1 alone. Taken together, these results establish Akt1 and Akt2 as key mediators of TNBC metastasis that may be targeted to prevent the spread of non-responsive disease.
Developing novel therapeutic strategies for TNBC patients who have RD after administration of NAC is vital to increasing patient survival. Improving current treatment regimens and reducing metastatic spread are two potential approaches that may prove advantageous. Pharmacological activation of AMPK with FND-4b suppressed TNBC growth, and future work could evaluate how adding AMPK activators to current NAC regimens impacts patient outcomes. AMPKα1 knockdown did not induce radiosensitization, but silencing Akt1 did potentiate radiation-induced apoptosis. Moreover, MK-2206 treatment reduced Akt1 expression and promoted synergistic apoptosis induction after radiotherapy. Further studies should determine how Akt1 suppression affects outcomes of TNBC patients with RD after receiving NAC. Finally, silencing Akt1 or Akt2 dramatically decreased TNBC lung metastasis. Knockdown of AMPKα isoforms did not reduce TNBC lung colonization, and combined inhibition of Akt1 and AMPKα1 did not suppress TNBC metastasis more than targeting Akt1 alone. Future studies should identify pharmacological agents that can decrease expression of Akt1 and Akt2 and evaluate their ability to reduce metastasis. Taken together, these studies provide potentially promising strategies to improve survival among TNBC patients whose tumors are refractory to current treatment modalities.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2020.329
Funding Information
This research was supported by the National Institutes of Health (NIH) grant T32 ES007266 (2016-2018), NIH grant R01 CA195573 (2018-2020), and Daphne's Legacy Breast Cancer Research Funds (2018-2020).
Recommended Citation
Johnson, Jeremy Andrew, "Examining the Role of Metabolic Pathways as Therapeutic Modalities for Triple Negative Breast Cancer" (2020). Theses and Dissertations--Toxicology and Cancer Biology. 32.
https://uknowledge.uky.edu/toxicology_etds/32
