Inhibition of IκB Kinase Is a Potential Therapeutic Strategy to Circumvent Resistance to Epidermal Growth Factor Receptor Inhibition in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) remains as an intractable malignancy with limited therapeutic targets. High expression of epidermal growth factor receptor (EGFR) has been associated with a poor prognosis of TNBC; however, EGFR targeting has failed with unfavorable clinical outcomes. Here, we performed a combinatorial screening of fifty-five protein kinase inhibitors with the EGFR inhibitor gefitinib in the TNBC cell line MDA-MB-231 and identified the IκB kinase (IKK) inhibitor IKK16 as a sensitizer of gefitinib. Cell viability and clonogenic survival assays were performed to evaluate the antiproliferative effects of the gefitinib and IKK16 (Gefitinib + IKK16) combination in TNBC cell lines. Western blot analyses were also performed to reveal the potential mode of action of this combination. In addition, next-generation sequencing (NGS) analysis was performed in Gefitinib+IKK16-treated cells. The Gefitinib+IKK16 treatment synergistically reduced cell viability and colony formation of TNBC cell lines such as HS578T, MDA-MB-231, and MDA-MB-468. This combination downregulated p-STAT3, p-AKT, p-mTOR, p-GSK3ß, and p-RPS6. In addition, p-NF-κB and the total NF-κB were also regulated by this combination. Furthermore, NGS analysis revealed that NF-κB/RELA targets including CCL2, CXCL8, EDN1, IL-1ß, IL-6, and SERPINE1 were further reduced and several potential tumor suppressors, such as FABP3, FADS2, FDFT1, SEMA6A, and PCK2, were synergistically induced by the Gefitinib-+IKK16 treatment. Taken together, we identified the IKK/NF-κB pathway as a potential target in combination of EGFR inhibition for treating TNBC.

Potentiating Therapeutic Effects of Epidermal Growth Factor Receptor Inhibition in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subset of breast cancer with aggressive characteristics and few therapeutic options. The lack of an appropriate therapeutic target is a challenging issue in treating TNBC. Although a high level expression of epidermal growth factor receptor (EGFR) has been associated with a poor prognosis among patients with TNBC, targeted anti-EGFR therapies have demonstrated limited efficacy for TNBC treatment in both clinical and preclinical settings. However, with the advantage of a number of clinically approved EGFR inhibitors (EGFRis), combination strategies have been explored as a promising approach to overcome the intrinsic resistance of TNBC to EGFRis. In this review, we analyzed the literature on the combination of EGFRis with other molecularly targeted therapeutics or conventional chemotherapeutics to understand the current knowledge and to provide potential therapeutic options for TNBC treatment.

Dual Inhibition of AKT and MEK Pathways Potentiates the Anti-Cancer Effect of Gefitinib in Triple-Negative Breast Cancer Cells.

There is an unmet medical need for the development of new targeted therapeutic strategies for triple-negative breast cancer (TNBC). With drug combination screenings, we found that the triple combination of the protein kinase inhibitors (PKIs) of the epidermal growth factor receptor (EGFR), v-akt murine thymoma viral oncogene homolog (AKT), and MAPK/ERK kinase (MEK) is effective in inducing apoptosis in TNBC cells. A set of PKIs were first screened in combination with gefitinib in the TNBC cell line, MDA-MB-231. The AKT inhibitor, AT7867, was identified and further analyzed in two mesenchymal stem-like (MSL) subtype TNBC cells, MDA-MB-231 and HS578T. A combination of gefitinib and AT7867 reduced the proliferation and long-term survival of MSL TNBC cells. However, gefitinib and AT7867 induced the activation of the rat sarcoma (RAS)/ v-raf-1 murine leukemia viral oncogene homolog (RAF)/MEK/ extracellular signal-regulated kinase (ERK) pathway. To inhibit this pathway, MEK/ERK inhibitors were further screened in MDA-MB-231 cells in the presence of gefitinib and AT7867. As a result, we identified that the MEK inhibitor, PD-0325901, further enhanced the anti-proliferative and anti-clonogenic effects of gefitinib and AT7867 by inducing apoptosis. Our results suggest that the dual inhibition of the AKT and MEK pathways is a novel potential therapeutic strategy for targeting EGFR in TNBC cells.

Anaplastic lymphoma kinase inhibitor NVP­TAE684 suppresses the proliferation of human pancreatic adenocarcinoma cells.

Anaplastic lymphoma kinase (ALK) is known to be an important therapeutic target in various types of cancer. NVP­TAE684, a well­known inhibitor of ALK, was revealed to exert antitumor effects in several different malignancies. However, the molecular mechanisms responsible for these antitumor effects in cancer cells, including pancreatic adenocarcinoma cells, remain unknown. In the present study, NVP­TAE684 was investigated for its antitumor effects towards pancreatic adenocarcinoma cells. MTT assay, western blot analysis, flow cytometry, caspase­3/7 activity assay and Trypan blue exclusion assay were used and it was revealed that NVP­TAE684 suppressed the proliferation of seven human pancreatic adenocarcinoma cell lines (AsPC­1, Panc­1, MIA PaCa­2, Capan­1, CFPAC­1, Colo­357 and BxPC­3), and significantly increased G2/M arrest and apoptotic cell death. Furthermore, NVP­TAE684 inhibited the phosphorylation of ALK at Y1604, as well as that of downstream mediators such as AKT (S473) and ERK1/2 (Y202/T204). Notably, knocking down ALK with siRNAs also decreased proliferation and promoted G2/M arrest and apoptosis. Furthermore, inhibition of ALK with NVP­TAE684 or siRNA synergistically enhanced gemcitabine­induced cell death by inducing apoptosis. In conclusion, the findings of the present study indicated that NVP­TAE684 exerted its antitumor effects by inducing G2/M arrest and apoptosis via the inhibition of the ALK signaling pathway, and suggests its potential use as an antitumor agent against pancreatic adenocarcinoma.

Ribosomal Protein S6: A Potential Therapeutic Target against Cancer?

Ribosomal protein S6 (RPS6) is a component of the 40S small ribosomal subunit and participates in the control of mRNA translation. Additionally, phospho (p)-RPS6 has been recognized as a surrogate marker for the activated PI3K/AKT/mTORC1 pathway, which occurs in many cancer types. However, downstream mechanisms regulated by RPS6 or p-RPS remains elusive, and the therapeutic implication of RPS6 is underappreciated despite an approximately half a century history of research on this protein. In addition, substantial evidence from RPS6 knockdown experiments suggests the potential role of RPS6 in maintaining cancer cell proliferation. This motivates us to investigate the current knowledge of RPS6 functions in cancer. In this review article, we reviewed the current information about the transcriptional regulation, upstream regulators, and extra-ribosomal roles of RPS6, with a focus on its involvement in cancer. We also discussed the therapeutic potential of RPS6 in cancer.

Inhibition of RPTOR overcomes resistance to EGFR inhibition in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) cells frequently exhibit activated growth factor signaling and resistance to inhibitors for epidermal growth factor receptor (EGFR), despite the overexpression of EGFR protein, and this is associated with a malignant behavior and a poor prognosis. In this study, to elucidate the underlying mechanisms of resistance to EGFR inhibitor and identify inhibitors that exert a synergistic effect with EGFR inhibition, we examined the inhibitory effects of selected protein kinase inhibitors (PKIs) in combination with gefitinib on the viability of a mesenchymal stem-like (MSL) subtype TNBC cell line. MK­2206, an AKT inhibitor, and a group of mammalian target of rapamycin (mTOR) inhibitors were found to exert synergistic lethal effects in combination with gefitinib in MDA­MB­231 cells. The combination of gefitinib/MK­2206 exerted a prominent synergistic lethal effect in an MTT cell viability assay and a growth inhibitory effect in a long-term colony-forming assay in 2 MSL subtype TNBC cell lines (MDA­MB­231 and HS578T) and one basal-like (BL) subtype TNBC cell line (MDA­MB-468). Gefitinib/MK­2206 treatment synergistically decreased the mTOR signaling target substrates along with the downregulation of ribosomal protein S6 (RPS6), a marker of cell proliferation and target substrate of the AKT-mTOR signaling pathway. In addition, gefitinib markedly reduced the viability of MDA­MD­231 and HS578T cells when regulatory-associated protein of mTOR (RPTOR) was suppressed by siRNA-based knockdown (KD). These results thus suggest that RPTOR mediates, at least partially, the resistance to EGFR inhibition in TNBC cells. Therefore, targeting the mTOR complex 1 (mTORC1) pathway may be a potential strategy for the treatment of EGFR-resistant TNBC.

Silencing of NRF2 Reduces the Expression of ALDH1A1 and ALDH3A1 and Sensitizes to 5-FU in Pancreatic Cancer Cells.

Pancreatic cancer remains an intractable cancer with a poor five-year survival rate, which requires new therapeutic modalities based on the biology of pancreatic oncogenesis. Nuclear factor E2 related factor-2 (NRF2), a key cytoprotective nuclear transcription factor, regulates antioxidant production, reduction, detoxification and drug efflux proteins. It also plays an essential role in cell homeostasis, cell proliferation and resistance to chemotherapy. We aimed to evaluate the possibility that modulation of NRF2 expression could be effective in the treatment of pancreatic cancer cells. We investigated whether the depletion of NRF2 by using small interfering RNAs (siRNAs) is effective in the expression of biomarkers of pancreatic cancer stemness such as aldehyde dehydrogenase 1 family, member A1 (ALDH1A1) and aldehyde dehydrogenase 3 family, member A1 (ALDH3A1). NRF2 knockdown markedly reduced the expression of NRF2 and glutamate-cysteine ligase catalytic subunit (GCLC) in cell lines established from pancreatic cancers. NRF2 silencing also decreased the ALDH1A1 and ALDH3A1 expression. Furthermore, this NRF2 depletion enhanced the antiproliferative effects of the chemotherapeutic agent, 5-fluorouracil (5-FU) in pancreatic cancer cells.