Mechanisms of acquired resistance to insulin-like growth factor 1 receptor inhibitor in MCF-7 breast cancer cell line.

The purpose of this study was to clarify the mechanism of acquired resistance to the insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase inhibitor NVP-AEW541. We developed an acquired resistant model by continuously exposing MCF-7 breast cancer cells to NVP-AEW541 (MCF-7-NR). MCF-7 and MCF-7-NR were comparatively analyzed for cell signaling and cell growth. While phosphorylation of Akt was completely inhibited by 3 µM NVP-AEW541 in both MCF-7 and MCF-7-NR, phosphorylation of S6K remained high only in MCF-7-NR, suggesting a disconnection between Akt and S6K in MCF-7-NR. Consistently, the mTOR inhibitor everolimus inhibited phosphorylation of S6K and cell growth equally in both lines. Screening of both lines for phosphorylation of 42 receptor tyrosine kinases with and without NVP-AEW541 showed that Tyro3 phosphorylation remained high only in MCF-7-NR. Protein expression of Tyro3 was found to be higher in MCF-7-NR than in MCF-7. Gene silencing of Tyro3 using siRNA resulted in reduced cell growth and cyclin D1 expression in both lines. While Tyro3 expression was inhibited by NVP-AEW541 and everolimus in MCF-7, it was reduced only by everolimus in MCF-7-NR. These findings suggested that cyclin D1 expression was regulated in a S6K/Tyro3-dependent manner in both MCF-7 and MCF-7-NR, and that the disconnection between IGF-1R/Akt and S6K may enable MCF-7-NR to keep cyclin D1 high in the presence of NVP-AEW541. In summary, acquired resistance to NVP-AEW541 appears to result from IGF-1R/Akt-independent activation of S6K and expression of Tyro3 and cyclin D1.

Excessive MET signaling causes acquired resistance and addiction to MET inhibitors in the MKN45 gastric cancer cell line.

The clinical efficacy of MET tyrosine kinase inhibitors (MET-TKIs) is hindered by the emergence of acquired resistance, presenting an obstacle to drug discovery. To clarify the mechanisms underlying acquired resistance to MET-TKIs, we established resistance models by continuous exposure of the MET-amplified gastric cancer cell line MKN45 to MET-TKIs, PHA665752 (MKN45-PR) and GSK1363089 (MKN45-GR). Baseline expression and phosphorylation of MET were elevated in MKN45-PR and MKN45-GR compared to MKN45 cells, and higher concentrations of MET-TKIs were required to inhibit MET phosphorylation compared to parental cells. Alterations in MET previously associated with resistance to MET-TKIs were observed in resistant cells, including elevated MET copy number, observed in both resistant lines compared to MKN45 cells, and the Y1230H mutation, detected in MKN45-PR cells. Notably, the growth of resistant lines was lower in the absence of MET-TKIs, suggesting "addiction" to inhibitors. While MKN45-PR cells exhibited a higher S-phase fraction in the absence of PHA665752, bromodeoxyuridine (BrdU) uptake was identical. Baseline phosphorylation of ATR, Chk1 and p53 and p21(waf1/Cip1) expression was higher in MKN45-PR compared to MKN45 cells, and levels were reduced to those observed in untreated MKN45 cells following PHA665752 treatment. Furthermore, targeted knockdown of MET enhanced the growth of MKN45-PR cells. These findings suggest that alterations in MET leading to acquired MET-TKI resistance, may cause excessive MET signaling, subsequent replication stress and DNA damage response, and intra-S-phase arrest in the absence of MET-TKIs. Thus, partial MET inhibition is necessary for resistant cells to proliferate, a phenomenon we refer to as MET-TKI "addiction".

Revisiting the IGF-1R as a breast cancer target.

The type I insulin-like growth factor-1 receptor is a well-described target in breast cancer and multiple clinical trials examining insulin-like growth factor-1 receptor have been completed. Unfortunately, monoclonal antibodies and tyrosine kinase inhibitors targeting insulin-like growth factor-1 receptor failed in phase III breast clinical trials for several reasons. First, insulin-like growth factor-1 receptor antibody therapy resulted in hyperglycemia and metabolic syndrome most likely due to disruption of insulin-like growth factor-1 homeostasis and subsequent growth hormone elevation. Growth hormone elevation induces insulin resistance, hence a subsequent elevation of insulin and the potential for activation of insulin receptor. Second, the insulin-like growth factor-1 receptor and insulin receptor are highly homologous in amino acid sequence, structure, and function. These two receptors bind insulin, insulin-like growth factor-1 and insulin-like growth factor-2, to regulate glucose uptake and other cellular functions. Hybrid receptors composed of one chain of insulin-like growth factor-1 receptor and insulin receptor also participate in signaling. Third, since all the monoclonal antibodies were specific for insulin-like growth factor-1 receptor, any pathophysiologic role for insulin receptor was not inhibited. While the insulin-like growth factor-1 receptor tyrosine kinase inhibitors effectively inhibited both insulin-like growth factor-1 receptor and insulin receptor, these drugs are not being further developed likely due to their metabolic toxicities. Insulin-like growth factor-1/2 neutralizing antibodies are still being studied in early phase clinical trials. Perhaps a more comprehensive strategy of targeting the insulin-like growth factor-1 receptor network would be successful. For example, targeting receptor, ligand and downstream signaling molecules such as phosphatidylinositol 3'-kinase or particularly the insulin receptor substrate adapter proteins might result in a complete blockade of insulin-like growth factor-1 receptor/insulin receptor biological functions.

TYRO3 as a potential therapeutic target in breast cancer.

AIM: We evaluated the potential of TYRO3 as a therapeutic target in various types of breast cancer cell lines. MATERIALS AND METHODS: The effects of TYRO3-knockdown by small interfering RNA (siRNA) on proliferation, cell-cycle distribution, and cell signaling in four estrogen receptor (ER)-positive/HER2-non-amplified (luminal-type), two ER-negative/HER2-amplified (HER2-type), and two ER-negative/HER2-non-amplified (triple negative [TN]-type) cell lines were compared. RESULTS: Whereas TYRO3 knockdown induced the greatest proliferation suppression in luminal-type cells, and to a lesser extent in HER2-type cells, no proliferation inhibition was observed in TN-type cells. The TYRO3 siRNA-induced proliferation inhibition in luminal-type cells was observed in both estradiol (E2)-rich and -null conditions. The proliferation suppression was correlated with G0-G1/S cell-cycle arrest. Western blot analysis showed a decrease in phosphorylation of ERK1/2 or STAT3, and in cyclin D1 only in cell lines sensitive to TYRO3-knockdown. CONCLUSION: TYRO3 is a potential therapeutic target in breast cancer, particularly in luminal-type cells.

Regulation of MET kinase inhibitor resistance by copy number of MET in gastric carcinoma cells.

We previously established acquired resistant models for MET-tyrosine kinase inhibitors (TKIs) by continuously exposing the MET-amplified gastric cancer cell line MKN45 to MET-TKIs, PHA665752 (MKN45-PR), or GSK1363089 (MKN45-GR). We found resistant mechanisms caused by increased copy number of MET in both lines and Y1230H mutation in MKN45-PR. We also found that excessive MET signaling caused by these MET alterations resulted in intra-S-phase arrest in the absence of MET-TKIs, so that cells grew faster in the presence of MET-TKIs, a phenomenon referred to as "addiction." In this study, to investigate reversibility of the acquired resistance and "addiction" to MET-TKIs and their causative MET alterations, we sequentially cultured MKN45-PR and MKN45-GR in decreasing concentrations of MET-TKIs until they were able to grow in a drug-free condition. These "revertant" cell lines (designated MKN45-PR-RE and MKN45-GR-RE) were comparatively analyzed. Growth assay showed that both MKN45-PR-RE and MKN45-GR-RE partially lost the property of "addiction" to MET-TKIs. MKN45-GR-RE lost the property of resistance to GSK1363089, but MKN45-PR-RE retained resistance to PHA665752. Copy numbers and expression and phosphorylation of MET protein reduced in both MKN45-PR-RE and MKN45-GR-RE compared with MKN45-PR and MKN45-GR, respectively, but Y1230H mutation and biochemical resistance to PHA665752 remained in MKN45-PR-RE. The "addiction" to MET-TKIs appeared attributable to increased copy number, and the property and the MET alteration were reversible. The Y1230H mutation appeared enough in itself to keep cells resistant to MET-TKIs and was irreversible.

Inhibition of the mTOR/S6K signal is necessary to enhance fluorouracil-induced apoptosis in gastric cancer cells with HER2 amplification.

The purpose of this study was to explore the effect of trastuzumab in enhancing the activity of chemotherapeutic agents and the molecular basis of this effect. Two gastric cancer cell types with HER2 amplification, one sensitive (NCI­N87) and one insensitive (MKN-7) to trastuzumab, were tested for the effects of trastuzumab on cell growth and cell signaling using MTS assay and western blotting, respectively. Interaction between trastuzumab and chemotherapeutic agents (fluorouracil, doxorubicin, cisplatin and paclitaxel) was evaluated by the combination index (CI). Fluorouracil-induced apoptosis was evaluated using western blot for poly (ADP-ribose) polymerase (PARP). Trastuzumab decreased phosphorylation of S6K, showed synergistic effect with fluorouracil or doxorubicin, and increased fluorouracil-induced apoptosis in NCI-N87 cells, but not in MKN-7 cells. While the mTOR inhibitor everolimus enhanced fluorouracil-induced apoptosis in both HER2-amplified cell lines, this was not the case in the gastric cancer cell lines without HER2 amplification. Consistently, while the EGFR/HER2 inhibitor Cl-387,785 inhibited cell growth of MKN-7, this growth inhibition did not accompany decrease in phosphorylation of S6K, and the compound did not enhance fluorouracil-induced apoptosis. In summary, inhibition of the mTOR/S6K signal may be a key molecular event in enhancing fluorouracil-induced apoptosis specifically in gastric cancer cells with HER2 amplification. mTOR inhibitors may therefore be attractive alternative drugs in gastric cancers with HER2 amplification regardless of their sensitivity to trastuzumab.