MARCKS Is Marked in Combating Lung Cancer Growth and Acquired Resistance
Authors: Sekhar P. Reddy, Viswanathan Natarajan, Arkadiusz Z. Dudek
Journal: American Journal of Respiratory and Critical Care Medicine (2014)

Lung cancer is a leading cause of cancer-related deaths worldwide. Genetic mutations in protooncogenes, such as epidermal growth factor receptor (EGFR), phosphatidylinositol-3-kinase (PI3K), RAS, and others, have been implicated in human lung cancer. Selective targeting of these effector pathways using genetic and pharmacological approaches mitigates experimental lung cancer, whereas mutated forms lead to tumor growth in rodents. Clinical trials with EGFR tyrosine kinase inhibitors (EGFR-TKIs) have demonstrated significant efficacy in patients with lung cancer, especially those with tumors that have activating mutation in EGFR. Eventually, however, tumors acquire resistance to the EGFR-TKIs, and disease progresses in all patients (1). Thus, it is vital to understand fully the mechanisms of resistance and to devise novel strategies to combat it.

PI3K signaling plays a key role in promoting cell proliferation, survival, motility, and metabolism. Dysregulation of this pathway has been implicated in the induction and/or progression of many diseases, including several respiratory diseases such as asthma and chronic obstructive pulmonary disease (2). Furthermore, this pathway is aberrantly activated in human cancer and plays a critical role in both the initiation and progression of experimental non–small cell lung cancer (3,4). Intriguingly, PI3K signaling has been shown to play a key role in resistance to therapy with EGFR-TKIs, and targeting this pathway is considered a viable option in treating patients with lung cancer with acquired resistance to TKIs (5). PI3K-mediated effector functions occur predominantly through the AKT signaling pathway. Phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylinositol-3,4,5-trisphosphate (PIP3) are phosphoinositides associated with the cell membrane, and PI3K-mediated phosphorylation of PIP2 generates PIP3, which in turn binds to and stimulates AKT activity (5) (Figure 1). Thus, a better understanding of the mechanisms that modulate cellular PIP2/PIP3 pools and aberrant activation of PI3K/AKT signaling may provide additional strategies to combat lung diseases, including cancer.



Figure 1.

Myristoylated alanine-rich c kinase substrate (MARCKS)-regulated phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylinositol-3,4,5-trisphosphate (PIP3) balance in the modulation of phosphatidylinositol-3-kinase (PI3K)-AKT signaling pathway in ...



In this issue of the Journal, Chen and colleagues (pp. 1127–1138) provide compelling evidence that a membrane-associated protein, MARCKS (myristoylated alanine-rich C kinase substrate), is a novel regulator of PIP3 levels in cancer cells (6). They found that phosphorylation of MARCKS at its phosphorylation site domain (PSD) is critical for PI3K-mediated conversion of membrane-associated PIP2 to PIP3, which is essential for lung cancer cell growth, progression, and resistance to EGFR-TKIs mediated by AKT activation. Mechanistically, they show that mutation or deletion of PSD of MARCKS increases the availability of PIP2 pools to PI3K. Perhaps this finding could explain a mechanism underlying the elevated levels of PIP3 frequently observed in aggressive cancer cells (3,4). Experimentally, the study provides the first demonstration of phospho-MARCKS in promoting lung cancer cell growth and metastasis, as well as resistance to an EFGR-TKI, erlotinib, in a preclinical model.

MARCKS is ubiquitously expressed in various tissues and is primarily phosphorylated by protein kinase C and Rho kinase (7,8). On phosphorylation of Ser159 and Ser163, located in the PSD, phospho-MARCKS detaches from the cell membrane (9) (Figure 1) and translocates into the cytoplasm. This step permits the release of PIP2 pools in the plasma membrane to PI3K, resulting in PIP3 generation. PSD is critical for MARCKS function, as it electrostatically binds to and sequesters PIP2 in the plasma membrane (10). PKG-induced phosphatase dephosphorylates MARCKS in the cytoplasm, allowing its translocation and interaction with the cell membrane and PIP2 (8), suggesting a balance between phosphorylated and unphosphorylated MARCKS is critical to maintain homeostasis in normal cells. However, in a recent study, the authors have reported increased levels of phospho-MARCKS in clinical specimens of patients with lung cancer (11), suggesting an imbalance between phosphorylated and unphosphorylated MARCKS can lead to pathology. Importantly, in the present study, Chen and colleagues found increased levels of phospho-MARCKS as a predictor of poor survival rates among patients with lung cancer specifically receiving EGFR-TKIs. In a preclinical model, they show that 25-mer peptide targeting PSD of MARCKS (or MSP) (12) markedly reduces lung tumor growth and metastasis in mice (13). Importantly, this peptide acts synergistically with erlotinib and exhibits efficacy in the inhibition of tumor growth, metastasis, and erlotinib resistance (6). These effects are preferentially induced in human cancers, with a sustained activation of PI3K signaling in PTEN-deficient and PIK3CA-mutant cells, which otherwise are resistant to erlotinib, both in vitro and in vivo. It is noteworthy that although MPS peptide was initially effective in reducing the growth of cancer cells that were otherwise resistant to EGFR-TKIs, further studies are warranted to test whether these cell lines initially respond to MPS peptide but later develop resistance.

In another recent study, the authors have shown that a peptide corresponding to the N terminus (myristoylated N-terminal sequence [MANS]) of MARCKS, termed MANS peptide (14), reduced lung cancer metastasis but had no effect on tumor growth in a preclinical model (13). In contrast, MSP peptide diminished both PIP3 pools and phospho-MARCKS levels by specifically suppressing MARCKS PSD activity (6). The lack of an effect on lung tumor growth mediated by the MANS peptide, although it reduces MARCKS' phosphorylation, appears to be caused by its inability to bind and sequester PIP2. On the basis of this new evidence, the authors propose that MSP, through binding to both MARCKS-PSD and PIP2, reduces the availability of PIP2 pools for P13K, thereby diminishing both PIP3 levels and the activation of PI3K/AKT signaling (Figure 1). How MSP peptide reduces the phosphorylation of MARCKS, which is already phosphorylated in cancer cells, and the nature of the phosphatase or phosphatases involved in dephosphorylation of MARCKS in cancer cells remain unclear. Furthermore, cells require PIP3 for normal cellular functions, such as vesicle trafficking and survival, and long-term depletion of PIP3 via MPS sequestration of PIP2 may be detrimental to cellular homeostasis. Thus, devising strategies to target MPS peptide to tumor cells is likely a preferable choice for cancer treatment.

The observations made by Chen and colleagues, using clinical specimens and phenotypic and mechanistic studies, represent an important advancement in the development of a novel strategy targeting MARCKS to combat lung cancer progression and EGFR-TKI resistance in cancer cells without activating EGFR mutation. Further work needs to be performed in lung cancer cell lines with activating EGFR mutation, where EGFR-TKIs are mostly used in clinical practice. Addressing with MSP resurgence of resistance to EGFR inhibition in this setting may have profound clinical implications. In this context, therapeutic inhibition of MARCKS activity, using the MPS peptide to suppress tumor cell growth and metastasis, as well as acquired drug resistance, may become a novel option to improve the survival of patients with lung cancer, and only when this is demonstrated could it warrant prospective clinical investigation. In addition, several inhibitors targeting the PI3K pathway are currently being investigated in preclinical and clinical studies (3,4), and targeting MARCKs may enhance and increase specificity to these agents in the inhibition of the PI3K pathway in lung cancer.