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Despite improvements in clinical outcomes of patients with colorectal cancer (CRC) over the past decades, prognosis remains poor, with a 5-year survival rate of less than 10 %. Fluoropyrimidine-based regimens have defined the standard of care for metastatic disease. However, with the identification of different genetic alterations involved in tumor cell signaling pathways, understanding the molecular pathogenesis of CRC has become of increasing importance in order to select personalized targeted therapies from which patients may potentially benefit. RAS mutations have proven to predict response to anti-epidermal growth factor receptor treatments. Additional hypothetical biomarkers of response, such as BRAF, PI3KCA, and PTEN, are under evaluation in ongoing clinical trials. In this article, we seek to review the recent development of novel drugs aimed to target these molecular aberrations and their impact on metastatic CRC patients.

Energy- and nutrient-sensing proteins such as AMPK, mTOR and S6K1 are now recognized as novel regulators of mitotic completion in proliferating cells. We investigated the cellular distribution of the Ser2481 autophosphorylation of mTOR, which directly monitors mTORC-specific catalytic activity, during mammalian cell mitosis and cytokinesis. Automated immunofluorescence experiments in human carcinoma cell lines revealed that phospho-mTOR (Ser2481) exhibited profound spatial and temporal dynamics during cell division. Phospho-mTOR (Ser2481) was strikingly enriched in mitotic cells, and in prophase, bright phospho-mTOR (Ser2481) staining could be clearly observed among condensed chromosomes. Phospho-mTOR (Ser2481) then redistributes from diffuse cytosolic staining that partially colocalizes with the mitotic spindle during the early phases of mitosis to the furrow at the onset of cytokinesis. Like the bona fide chromosomal passenger proteins (CPPs) INCENP and Aurora B, phospho-mTOR (Ser2481) displayed noteworthy accumulation in the central spindle midzone and the midbody regions, which persisted during the furrowing process. Accordingly, double-staining experiments confirmed that phospho-mTOR (Ser2481) largely colocalized with CCPs in the midbodies. The CPP-like mitotic localization of phospho-mTOR (Ser2481) was fully prevented by the microtubule-depolymerizing drug nocodazole; mitotic traveling of phospho-mTOR (Ser2481) to the midbody during telophase and cytokinesis, where it appears to be integrated into the CPP-driven cytokinetic machinery, may therefore require dynamic microtubules. Although the Ser2448-phosphorylated form of mTOR was also found at high levels during M-phase in human cancer cells, we failed to observe a significant association of phospho-mTOR (Ser2448) with CCP-positive mitotic and cytokinetic structures. Our findings add phospho-mTOR (Ser2481) to the growing list of phospho-active forms of proteins belonging to the AMPK/mTOR/S6K1 signaling axis that reside at the mitotic and cytokinetic apparatus. Future studies should elucidate how the specific ability of phospho-mTOR (Ser2481) to spatially and temporally couple to the cleavage furrow and midbody region as a CPP-like protein can signal to or from adjacent signaling complexes and/or with the basic machinery of cell abscission.

Autophagy, an evolutionary conserved catabolic process whereby cells generate energy and building blocks by promoting large-scale recycling of cytoplasmic macromolecules and organelles, represents a novel drug-targetable molecular mechanism underlying de novo (primary) refractoriness of HER2 gene-amplified breast cancer (BC) to HER2 inhibition. JIMT-1 cells, which were obtained from a HER2-positive BC patient that rapidly progressed on trastuzumab (Herceptin™) ab initio and that show cross-resistance to multiple HER1/2 inhibiting drugs including lapatinib (Tykerb™), were found to constitutively exhibit an enhanced autophagic vesicle content as assessed by immunoblotting of endogenous ATG8/LC3 lipidation and confocal imaging of the recruitment of ATG8/LC3 to autophagic vesicles. A significant decrease in the expression status of the specific autophagy receptor p62/SQSTM1 -a protein selectively degraded by autophagy- confirmed further a constitutive activation of the autophagic flux in trastuzumab-refractory JIMT-1 cells. When the Human Autophagy RT2 Profiler™ PCR Array was employed to profile the expression of 84 genes involved in autophagy, ATG12 was the most differentially up-regulated gene in JIMT-1 cells (>10-fold) as compared with trastuzumab-responsive SKBR3 cells. Upon collection of the transcriptional profile of the ATG12 gene across two sets of > 50 widely used BC cell lines, HER2+ BC cells with well-established de novo resistance to trastuzumab were characterized by expressing significantly higher levels of ATG12. When lentiviral-delivered small hairpin RNA was employed to stably & specifically knock-down ATG12 gene, JIMT-1 ATG12-shRNA cells were more significantly growth-inhibited by trastuzumab (up to 5-fold) than parental JIMT-1 cells. Moreover, the half-maximal inhibitory concentration (IC50) values for the small-molecule HER1/2 Tyrosine Kinase Inhibitors gefitinib, erlotinib and lapatinib were drastically reduced by up to 10 times in response to ATG12 gene silencing. Knockdown of autophagy-regulatory genes other than ATG12 (e.g. ATG5, ATG8/LC3B) similarly suppressed refractoriness to trastuzumab as well as co-existing resistance to other HER1/2-targeted agents. Knockdown of autophagy-specific genes, however, did not alter basal sensitivity of JIMT-1 cells to multiple cytotoxics including doxorubicin, cisplatin, paclitaxel and vinorelbine. When lysosomal degradation was pharmacologically inhibited by using the antimalarial lysosomotropic drug chloroquine we found a massive accumulation of abnormal autophagolysosomes that promoted synergistic re-sensitization of JIMT-1 cells to the growth inhibitory and apoptotic activity of trastuzumab. In summary, autophagy-specific genes appear to play a potent protective role against HER2 inhibiting drugs currently in use. Given that genetic and pharmacological targeting of autophagy was found to be detrimental to intrinsic BC refractoriness to HER2−targeted therapies, our current findings may provide rationale for novel, chloroquine-based therapeutic approaches aimed to circumvent primary-resistance and potentiate the efficacy of both trastuzumab and lapatinib in patients treated for HER2−positive BC disease. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-14.

Primary resistance of HER2 gene-amplified breast carcinomas (BC) to HER-targeted therapies can be explained in terms of overactive HER2-independent downstream pro-survival pathways. We here confirm that constitutive overexpression of Inhibitor of Apoptosis (IAP) survivin is indispensable for survival of HER2-positive BC cells with intrinsic cross-resistance to multiple HER1/2 inhibitors. The IC₅₀ values for the HER1/2 Tyrosine Kinase Inhibitors (TKIs) gefitinib, erlotinib and lapatinib were up to 40-fold higher in trastuzumab-unresponsive JIMT-1 cells than in trastuzumab-naïve SKBR3 cells. ELISA-based and immunoblotting assays demonstrated that trastuzumab-refractory JIMT-1 cells constitutively expressed ~ 4 times more survivin protein than trastuzumab-responsive SKBR3 cells. In response to trastuzumab, JIMT-1 cells accumulated ~10 times more survivin than SKBR3 cells. HER1/2 TKIs failed to down-regulate survivin expression in JIMT-1 cells whereas equimolar doses of HER1/HER2 TKIs drastically depleted survivin protein in SKBR3 cells. ELISA-based detection of histone-associated DNA fragments confirmed that trastuzumab-refractory JIMT-1 cells were intrinsically protected against the apoptotic effects of HER1/2 TKIs. Of note, when we knocked-down survivin expression using siRNA and then added trastuzumab, cell proliferation and colony formation were completely suppressed in JIMT-1 cells. Our current findings may be extremely helpful to design successful combinatorial strategies aimed to circumvent the occurrence of de novo resistance to HER2-directed drugs using survivin antagonists.

In the previous issue of Breast Cancer Research, Jin and colleagues [1] demonstrated that the lipogenic enzyme fatty acid synthase (FASN) is phosphorylated when it is in complex with the tyrosine kinase receptor HER2. HER2-dependent activating phosphorylation of FASN was related to tumor progression and was sensitive to the dual HER1/HER2 tyrosine kinase inhibitor lapatinib [1]. Adding to knowledge of the therapeutic value of the phosphorylation status of FASN in HER2-driven breast cancer (BC), we now provide experimental evidence that the expression status of FASN in the extracellular milieu (that is, extracellular FASN) may function as a novel biomarker that distinctively predicts molecular functioning of lapatinib. AMP-activated protein kinase (AMPK)-activating drugs, by mimicking an elevated AMP/ATP ratio in BC cells, drastically augment the release of extracellular FASN in HER2-positive BC cells [2]. Lapatinib-induced deprivation of tumor cell energy activates AMPK to trigger an entire cascade of metabolic events, including suppression of FASN expression and activity [1,3]. We hypothesized that a differential ability to initiate AMPK-sensed metabolic stress responses may provide information about the efficacy of HER-targeting drugs via changes in the extracellular FASN status. Enzyme-linked immunosorbent assay (ELISA)-based quantitative analyses revealed that lapatinib treatment drastically enhanced extracellular FASN concentration (by at least 8.0-fold) (Figure ​(Figure1a).1a). Immunoblotting assessment of AMPK phosphorylation at Thr172 confirmed that lapatinib treatment induced a strong activation of AMPK (Figure ​(Figure1b).1b). A weak but detectable upregulation of PP-AMPKThr172 was observed upon treatment with gefitinib. Trastuzumab, cetuximab, and erlotinib - all of which are unable to promote FASN release - failed to activate AMPK. AMPK knockdown using short interfering RNA (siRNA) transfection [2] fully prevented lapatinib-induced FASN release (Figure S1). Immunoblotting and cell imaging analyses confirmed that FASN was depleted from the cytosol of lapatinib-treated HER2 overexpressors and accumulated in their extracellular milieu (Figure S1). Figure 1 Lapatinib's molecular functioning involves AMPK-dependent release of extracellular fatty acid synthase (FASN). (a) HER1 or HER2 inhibitors (or both) differentially regulate extracellular expression of FASN. FASN concentration in cell culture supernatants ... Treatment with lapatinib dramatically increased FASN release (by approximately 17 times) in lapatinib-responsive SKBR3 TzbR cells (Figure ​(Figure1c),1c), which were selected for long-term outgrowth in trastuzumab-containing culture medium. Extracellular FASN remained unaltered in response to trastuzumab or lapatinib in JIMT-1 BC cells, which exhibit de novo cross-refractoriness to multiple HER1/2-targeted therapies (Figure ​(Figure1c).1c). Lapatinib treatment significantly activated AMPK and promoted an enormous accumulation of extracellular FASN in lapatinib-hypersensitive MCF-7/HER2 cells (Figure ​(Figure1d).1d). Equimolar concentrations of lapatinib failed to activate AMPK or to alter FASN release into the extracellular milieu of MCF-7/HER2 LapR cells, which were obtained by continuously subculturing parental MCF-7/HER2 cells with high-dose lapatinib. High levels of circulating FASN can be found in peripheral blood of HER2-overexpressing BC patients [4]. The exclusive ability of lapatinib to actively promote the extracellular release of FASN via AMPK-sensed energy depletion in metabolically demanding HER2-driven BC cell growth might provide a molecular rationale to evaluate the predictive value of circulating FASN in HER2-positive BC patients receiving lapatinib.