Evidence is mounting that the occurrence of the CD44(pos)/CD24(neg/low) cell population, which contains potential breast cancer (BC) stem cells, could explain BC clinical resistance to HER2-targeted ...therapies. We investigated whether de novo refractoriness to the anti-HER2 monoclonal antibody trastuzumab (Tzb; Herceptin) may relate to the dynamic regulation of the mesenchymal CD44(pos)/CD24(neg/low) phenotype in HER2-positive BC. We observed that the subpopulation of Tzb-refractory JIMT-1 BC cells exhibiting CD44(pos)/CD24(neg/low)-surface markers switched with time. Low-passage JIMT-1 cell cultures were found to spontaneously contain approximately 10% of cells bearing the CD44(pos)/CD24(neg/low) immunophenotype. Late-passage (>60) JIMT-1 cultures accumulated approximately 80% of CD44(pos)/CD24(neg/low) cells and closely resembled the CD44(pos)/CD24(neg/low)-enriched ( approximately 85%) cell population constitutively occurring in HER2-negative MDA-MB-231 mesenchymal BC cells. Dynamic expression of mesenchymal markers was not limited to CD44/CD24 because high-passages of JIMT-1 cells exhibited also reduced expression of the HER2 protein and over-secretion of pro-invasive/metastatic chemokines and metalloproteases. Accordingly, late-passage JIMT-1 cells displayed an exacerbated migratogenic phenotype in plastic, collagen, and fibronectin substrates. Intrinsic genetic plasticity to efficiently drive the emergence of the CD44(pos)/CD24(neg/low) mesenchymal phenotype may account for de novo resistance to HER2 targeting therapies in basal-like BC carrying HER2 gene amplification.
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.
The restoration of pluripotency circuits by the reactivation of endogenous stemness factors, such as SOX2, may provide a new paradigm in cancer development. The tumoral stem cell reprogramming ...hypothesis, i.e., the ability of stemness factors to redirect normal and differentiated tumor cells toward a less-differentiated and stem-like state, adds new layers of complexity to cancer biology, because the effects of such reprogramming may remain dormant until engaged later in response to (epi)genetic and/or (micro)environmental events. To test this hypothesis, we utilized an in vitro model of a SOX2-overexpressing cancer stem cell (CSC)-like cellular state that was recently developed in our laboratory by employing Yamanaka's nuclear reprogramming technology in the estrogen receptor α (ERα)-positive MCF-7 breast cancer cell line. Despite the acquisition of distinct molecular features that were compatible with a breast CSC-like cellular state, such as strong aldehyde dehydrogenase activity, as detected by ALDEFLUOR, and overexpression of the SSEA-4 and CD44 breast CSC markers, the tumor growth-initiating ability of SOX2-overexpressing CSC-like MCF-7 cells solely occurred in female nude mice supplemented with estradiol when compared with MCF-7 parental cells. Ser118 phosphorylation of estrogen receptor α (ERα), which is a pivotal integrator of the genomic and nongenomic E
2
/ERα signaling pathways, drastically accumulated in nuclear speckles in the interphase nuclei of SOX2-driven CSC-like cell populations. Moreover, SOX2-positive CSC-like cells accumulated significantly higher numbers of actively dividing cells, and the highest levels of phospho-Ser118-ERα occurred when chromosomes lined up on a metaphase plate. The previously unrecognized link between E
2
/ERα signaling and SOX2-driven stem cell circuitry may significantly impact our current understanding of breast cancer initiation and progression, i.e., SOX2 can promote non-genomic E
2
signaling that leads to nuclear phospho-Ser118-ERα, which ultimately exacerbates genomic ER signaling in response to E
2
. Because E
2
stimulation has been recently shown to enhance breast tumor-initiating cell survival by downregulating miR-140, which targets SOX2, the establishment of a bidirectional cross-talk interaction between the stem cell self-renewal regulator, SOX2, and the local and systemic ability of E
2
to increase breast CSC activity may have profound implications for the development of new CSC-directed strategies for breast cancer prevention and therapy.
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.
Characterization of the molecular function of complex phenols naturally present in extra virgin olive oil (EVOO) against the HER2-gene amplified JIMT-1 cell line, a unique breast cancer model that ...inherently exhibits cross-resistance to multiple HER1/2-targeted drugs including trastuzumab, gefitinib, erlotinib and lapatinib, may underscore innovative cancer molecules with novel therapeutic targets because they should efficiently circumvent de novo resistance to HER1/2 inhibitors in order to elicit tumoricidal effects. We identified pivotal signaling pathways associated with the efficacy of crude phenolic extracts (PEs) obtained from 14 monovarietals of Spanish EVOOs. i) MTT-based cell viability and HPLC coupled to time-of-flight (TOF) mass spectrometry assays revealed that anti-cancer activity of EVOO PEs positively correlated with the phenolic index (i.e., total content of phenolics) and with a higher presence of the complex polyphenols secoiridoids instead of lignans. ii) Genome-wide analyses using 44 K Agilent's whole human arrays followed by Gene Set Enrichment Analysis (GSEA)-based screening of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database revealed a differential modulation of the JIMT-1 transcriptome at the level of the cell cycle and p53 pathways. EVOO PEs differentially impacted the expression status of stress-sensing, G2-M check-point-related GADD45 genes and of p53-related CDKN1A, CDKN1C and PMAIP-1 genes. iii) Cell cycle and fluorescence microscopy analyses confirmed that secoiridoid-rich EVOO PE inhibited mitosis to promote G2-M cell cycle arrest. This was accompanied with the appearance of diffuse, even DNA staining with γH2AX and pan-nuclear hyperacetylation of Histone H3 at Lysine 18. iv) Semi-quantitative Signaling Node Multi-Target ELISAs determined that secoiridoid-rich EVOO PE drastically activated the mitogen-activated protein kinases MEK1 and p38 MAPK, a GADD45-related kinase involved in Histone H3 acetylation. Secoiridoids, a family of complex polyphenols characteristic of Oleaceae plants, appear to permit histones to remain in hyperacetylated states and through the resulting alterations in gene regulation to reduce mitotic viability and metabolic competence of breast cancer cells inherently refractory to HER-targeting therapies ab initio. Oleaceae secoiridoids could provide a valuable phytochemical platform for the design of more pharmacologically active second-generation phytopharmaceutical anti-breast cancer molecules with a unique mode of action.
CD24, a mucin-like adhesion molecule that enhances the metastatic potential of malignant cells, has been suggested to be a marker of poor prognosis in breast carcinomas. The tumor-initiating ...potential of CD44posCD24pos cell populations has been recently recognized and, accordingly, distant metastases are largely composed of CD24-positive cells in breast cancer patients refractory to treatment. Therefore, new therapeutic strategies aimed at down-regulating CD24 may negatively regulate the dissemination of tumor cells and formation of metastasis. Here, we reveal that suppression of CD24 protein expression is a crucial event in the molecular mechanisms underlying the growth-inhibitory effects of the anti-diabetic drug metformin in MDA-MB-468 triple-negative (basal-like) breast cancer cells. First, we confirmed that, among the different molecular classes of breast cancer, basal-like breast cancer cells were significantly more sensitive to the growth-inhibitory effects of metformin. Second, we observed a positive correlation between the growth inhibitory activity of metformin and the relative enrichment in cells bearing the CD44posCD24pos immunophenotype. Third, high-content indirect immunofluorescence imaging assays revealed that CD24 protein levels were drastically decreased in the presence of growth-inhibitory concentrations of metformin. Fourth, to preliminary assess the clinical relevance of metformin's anti-CD24 effects we took advantage of the recently developed ROCK online interface (http://rock.icr.ac.uk/), a publicly accessible portal that allows rapid integration of breast cancer functional and molecular profiling datasets. When we evaluated the impact of CD24 expression on distant metastasis-free survival (DMFS) in microarray gene expression breast cancer datasets, Kaplan-Meier survival analyses and log-rank tests comparing DMSF for CD24-high and CD24-low breast carcinomas revealed that patients with CD24-high tumors tended to have a shorter DMFS. These findings, altogether, suggest that the ability of metformin to suppress the oncogene, metastasis promoter and breast cancer stem cell marker CD24 may open a novel molecular avenue in the therapeutic management of highly-metastastic subgroups of triple-negative (basal-like) breast cancers naturally enriched with CD44posCD24pos tumor-initiating cell populations.
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.
Cancer cells expressing constitutively active phosphatidylinositol-3 kinase (PI3K) are proliferative regardless of the absence of insulin, and they form dietary restriction (DR)-resistant tumors in ...vivo. Because the binding of insulin to its receptors activates the PI3K/AKT/mammalian target of rapamycin (mTOR) signaling cascade, activating mutations in the PIK3CA oncogene may determine tumor response to DR-like pharmacological strategies targeting the insulin and mTOR pathways. The anti-diabetic drug metformin is a stereotypical DR mimetic that exerts its anti-cancer activity through a dual mechanism involving insulin-related (systemic) and mTOR-related (cell-autonomous) effects. However, it remains unclear whether PIK3CA-activating mutations might preclude the anti-cancer activity of metformin in vivo. To model the oncogenic PIK3CA-driven early stages of cancer, we used the clonal breast cancer cell line MCF10DCIS.com, which harbors the gain-of-function H1047R hot-spot mutation in the catalytic domain of the PI3KCA gene and has been shown to form DR-refractory xenotumors. To model PIK3CA-activating mutations in late stages of cancer, we took advantage of the isogenic conversion of a PIK3CA-wild-type tumor into a PIK3CA H1047R-mutated tumor using the highly metastatic colorectal cancer cell line SW48. MCF10DCIS.com xenotumors, although only modestly affected by treatment with oral metformin (approximately 40% tumor growth inhibition), were highly sensitive to the intraperitoneal (i.p.) administration of metformin, the anti-cancer activity of which increased in a time-dependent manner and reached >80% tumor growth inhibition by the end of the treatment. Metformin treatment via the i.p. route significantly reduced the proliferation factor mitotic activity index (MAI) and decreased tumor cellularity in MCF10DCIS.com cancer tissues. Whereas SW48-wild-type (PIK3CA+/+) cells rapidly formed metformin-refractory xenotumors in mice, ad libitum access to water containing metformin significantly reduced the growth of SW48-mutated (PIK3CAH1047R/+) xenotumors by approximately 50%. Thus, metformin can no longer be considered as a bona fide DR mimetic, at least in terms of anti-cancer activity, because tumors harboring the insulin-unresponsive, DR-resistant, PIK3CA-activating mutation H1047R remain sensitive to the anti-tumoral effects of the drug. Given the high prevalence of PIK3CA mutations in human carcinomas and the emerging role of PIK3CA mutation status in the treatment selection process, these findings might have a significant impact on the design of future trials evaluating the potential of combining metformin with targeted therapy.
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