Interferon/STAT1 and neuregulin signaling pathways are exploratory biomarkers of cetuximab (Erbitux®) efficacy in KRAS wild-type squamous carcinomas: a pathway-based analysis of whole human-genome microarray data from cetuximab-adapted tumor cell-line models.

KRAS mutation status is being used as the sole biomarker to predict therapeutic efficacy of cetuximab in metastatic colorectal cancer (mCRC). A significant number of mCRC patients with KRAS wild-type (WT) tumors, however, do not benefit from cetuximab. We are also lacking efficacy predictors in head and neck squamous cell carcinomas with an intact KRAS signaling and in non-small cell lung cancer in which KRAS mutations do not predict cetuximab efficacy. We recently established pre-clinical models of EGFR gene-amplified KRAS WT A431 squamous carcinoma cells chronically adapted to grow in the presence of cetuximab. We employed the ingenuity pathway analysis software to functionally interpret data from Agilent's whole human genome arrays in the context of biological processes, networks, and pathways. Cetuximab-induced activation of the interferon (IFN)/STAT1 appeared to switch from 'growth inhibitory' in acutely-treated cells to 'pro-survival' in chronically-adapted cells. Cetuximab treatment appeared to negatively select initially dominant IFN-sensitive clones and promoted selection of IFN- and cetuximab-refractory tumor clones constitutively bearing an up-regulated IFN/STAT1 signaling. High-levels of mRNAs coding for the EGFR ligands amphiregulin (AREG), epiregulin (EREG), and neuregulin-1/heregulin (NRG1) predicted for acute cetuximab's functioning. Chronic cetuximab, however, appeared to negatively select initially dominant AREG/EREG/NRG1-positive clones to promote selection of cetuximab-refractory clones exhibiting a knocked-down neuregulin signaling. Our current evolutionary mapping of the transcriptomic changes that occur during cetuximab-induced chronic blockade of EGFR/KRAS WT signaling strongly suggests that mRNAs coding for IFN/STAT1- and EGFR ligands-related genes can be evaluated as novel predictors of efficacy in KRAS WT squamous cancer patients being treated with cetuximab.

Pathway-focused proteomic signatures in HER2-overexpressing breast cancer with a basal-like phenotype: new insights into de novo resistance to trastuzumab (Herceptin).

Pioneering clinical studies in de novo refractoriness to the anti-HER2 monoclonal antibody trastuzumab have suggested that HER2 gene-amplification can take place also in a basal-like molecular background to generate basal/HER2+ tumors intrinsically resistant to trastuzumab. Here, we first investigated the unique histogenesis of the basal/HER2+ phenotype in breast carcinomas. The presence of basal CK5/CK6 cytokeratin expression in HER2+ tumors revealed a significant overlap in the histological features of HER2+/CK5/6+ and basal-like breast carcinomas. Basal/HER2+ tumors were typically poorly differentiated, high-grade invasive ductal carcinomas with large geographic necrosis, pushing margins of invasion, syncytial arrangement of tumor cells, ribbon- or festoon-like architecture, squamous metaplasia, stromal lymphocytic infiltrates, high mitotic index and strong p53 positivity. Secondly, we performed low-scale proteomic approaches in JIMT-1 cells, a unique model of HER2-gene amplified trastuzumab-resistant breast carcinoma with a basal-like phenotype, to develop biomarker signatures that may differentiate trastuzumab-responsive from non-responsive tumors. When applying antibody-based array technology to the extracellular milieu of trastuzumab-refractory JIMT-1 and trastuzumab-sensitive SKBR3 cell cultures, JIMT-1 cells were found to secrete higher amounts of several growth factors including amphiregulin, EGF, IGFBP-6, PDGF-AA, neurotrophins, TGFbeta and VEGF. Semi-quantitative signaling node multi-target sandwich ELISAs revealed that JIMT-1 cells drastically overactivate RelA, the prosurvival subunit of NF-kappaB as compared to trastuzumab-sensitive luminal/HER2+ SKBR3 cells. When simultaneously assessing the activation status of 42 receptor tyrosine kinases (RTK) using a human phospho-RTK array, JIMT-1 cells were found to constitutively display hyperactivation of the insulin-like growth factor-I receptor (IGF-1R). High-content immunofluorescence imaging revealed that activated IGF-1R mainly localized at focal adhesion-like structures in JIMT-1 cells. In vitro wound healing assays suggested that this functional reorganization of the JIMT-1 cytoskeletal reorganization may account for an exacerbated trastuzumab-refractory 'migratogenic' phenotype. Forthcoming studies should validate the notion that identification of basal-like immunophenotypes and/or basal-like molecular signatures within HER2+ breast carcinomas may provide rapid means to define subgroups of breast cancer patients likely to display resistance to trastuzumab ab initio.

Serine 2481-autophosphorylation of mammalian target of rapamycin (mTOR) couples with chromosome condensation and segregation during mitosis: confocal microscopy characterization and immunohistochemical validation of PP-mTOR(Ser2481) as a novel high-contrast mitosis marker in breast cancer core biopsies.

The prognostic abilities of breast cancer gene expression signatures are due mostly to the detection of proliferation activity. One of the strongest, yet simple and well-reproducible proliferation-associated prognostic factors is the mitotic activity index (MAI). However: a) counting mitotic figures is regarded by many histopathologists as cumbersome and time-consuming, and b) most available immunohistochemical markers are much weaker predictors than the MAI. We have investigated the spatio-temporal sub-cellular distribution of the Serine 2481-autophosphorylated form of mTOR (PP-mTOR(Ser2481)) during the G(1)/S-to-M-phase transition both in cultured cancer cells and in cancer tissue specimens. Using a high-resolution, automated confocal high-content imaging system, we observed that mitotic cells notably accumulated a distinct pattern of nuclear and cytoplasmic immunolabelings of PP-mTOR(Ser2481). Parallel experiments examining site-specific phosphorylation (i.e., Serine 10 and Serine 28) of the G(2)/M marker Histone H3 (PP-H3) revealed that PP-H3(Ser10/Ser28) staining efficiently detected mitotic cells from prophase until the beginning of anaphase, but not during late anaphase, telophase and cytokinesis. PP-mTOR(Ser2481) staining associated near and between separating chromosomes not only during early mitotic stages but also to the midzone and to midbody at ana/telophase through cytokinesis. We then evaluated the usefulness of PP-mTOR(Ser2481) immunostaining for improving the efficiency of mitotic counting using. Anti-PP-mTOR(Ser2481)-labeled mitotic figures (MFs) were easily seen and permitted a quick identification of mitotic hotspots in formalin-fixed cancer tissues, even at low magnification. Importantly, average mitotic counts were significantly higher when using PP-mTOR(Ser2481) staining than with the hematoxylin and eosin (H&E) protocol in breast cancer core biopsies. Mitotic count based on PP-mTOR(Ser2481) immunostaining increased tumor grade by one grade in 2 of 9 breast carcinomas. These findings warrant forthcoming studies to confirm both the accuracy and the prognostic value of PP-mTOR(Ser2481) as a novel high-contrast immunohistochemical mitosis marker in larger populations of human breast carcinomas.

Mitotic kinase dynamics of the active form of AMPK (phospho-AMPKalphaThr172) in human cancer cells.

When interrogating the activation status of AMP-activated protein kinase-measured as AMPKalpha(Thr172) phosphorylation-in tissue sections of human carcinomas and in cultured human cancer cells, the spatiotemporal dynamics of AMPK activity during the G(1)/S-to-M-phase transition strikingly resembles that of well-characterized "chromosomal passenger" proteins such as Aurora B, INCENP or Histone H3. The mitotic kinase behavior of the active form of AMPK may represent a candidate molecular link through which energy status directly influences tumorigenesis. A definitive elucidation of phospho-AMPKalpha(Thr172) in coordinating the chromosomal and cytoskeletal events of mitosis might radically amend our current perception of other AMPK-related diseases such as obesity, cardiac hypertrophy or accelerated aging syndromes.