Pang-Kuo Lo

CD49f and CD61 identify Her2/neu-induced mammary tumor-initiating cells that are potentially derived from luminal progenitors and maintained by the integrin–TGFβ signaling

Human epidermal growth factor receptor 2 (HER2)/Neu is overexpressed in 20–30% of breast cancers and associated with aggressive phenotypes and poor prognosis. For deciphering the role of HER2/Neu in breast cancer, mouse mammary tumor virus (MMTV)-Her2/neu transgenic mice that develop mammary tumors resembling human HER2-subtype breast cancer have been established. Several recent studies have revealed that HER2/Neu is overexpressed in and regulates self renewal of breast tumor-initiating cells (TICs). However, in the MMTV-Her2/neu transgenic mouse model, the identity of TICs remains elusive, despite previous studies showing supportive evidence for existence of TICs in Her2/neu-induced mammary tumors. Through systematic screening and characterization, we identified that surface markers CD49f, CD61 and ESA were aberrantly overexpressed in Her2-overexpressing mammary tumor cells. Analysis of these markers and CD24 detected anomalous expansion of the luminal progenitor population in preneoplastic mammary glands of Her2/neu transgenic mice, indicating that aberrant luminal progenitors originated in Her2-induced mammary tumors. The combined markers, CD49f and CD61, further delineated the CD49fhighCD61high-sorted fraction as a TIC-enriched population, which displayed increased tumorsphere formation ability, enhanced tumorigenicity both in vitro and in vivo and drug resistance to pacitaxel and doxorubicin. Moreover, the TIC-enriched population manifested increased transforming growth factor-β (TGFβ) signaling and exhibited gene expression signatures of stemness, TGFβ signaling and epithelial-to-mesenchymal transition. Our findings that self-renewal and clonogenicity of TICs were suppressed by pharmacologically inhibiting the TGFβ signaling further indicate that the TGFβ pathway is vital for maintenance of the TIC population. Finally, we showed that the integrin-β3 (CD61) signaling pathway was required for sustaining active TGFβ signaling and self-renewal of TICs. We for the first time developed a technique to highly enrich TICs from mammary tumors of Her2/neu transgenic mice, unraveled their properties and identified the cooperative integrin-β3–TGFβ signaling axis as a potential therapeutic target for HER2-induced TICs.

Deactivation of Akt by a small molecule inhibitor targeting pleckstrin homology domain and facilitating Akt ubiquitination

The phosphatidylinositol-3,4,5-triphosphate (PIP3) binding function of pleckstrin homology (PH) domain is essential for the activation of oncogenic Akt/PKB kinase. Following the PIP3-mediated activation at the membrane, the activated Akt is subjected to other regulatory events, including ubiquitination-mediated deactivation. Here, by identifying and characterizing an allosteric inhibitor, SC66, we show that the facilitated ubiquitination effectively terminates Akt signaling. Mechanistically, SC66 manifests a dual inhibitory activity that directly interferes with the PH domain binding to PIP3 and facilitates Akt ubiquitination. A known PH domain-dependent allosteric inhibitor, which stabilizes Akt, prevents the SC66-induced Akt ubiquitination. A cancer-relevant Akt1 (e17k) mutant is unstable, making it intrinsically sensitive to functional inhibition by SC66 in cellular contexts in which the PI3K inhibition has little inhibitory effect. As a result of its dual inhibitory activity, SC66 manifests a more effective growth suppression of transformed cells that contain a high level of Akt signaling, compared with other inhibitors of PIP3/Akt pathway. Finally, we show the anticancer activity of SC66 by using a soft agar assay as well as a mouse xenograft tumor model. In conclusion, in this study, we not only identify a dual-function Akt inhibitor, but also demonstrate that Akt ubiquitination could be chemically exploited to effectively facilitate its deactivation, thus identifying an avenue for pharmacological intervention in Akt signaling.

Electrospun fibrous scaffolds promote breast cancer cell alignment and epithelial-mesenchymal transition.

In this work we created electrospun fibrous scaffolds with random and aligned fiber orientations in order to mimic the three-dimensional structure of the natural extracellular matrix (ECM). The rigidity and topography of the ECM environment have been reported to alter cancer cell behavior. However, the complexity of the in vivo system makes it difficult to isolate and study such extracellular topographical cues that trigger cancer cells response. Breast cancer cells were cultured on these fibrous scaffolds for 3-5 days. The cells showed elongated spindle-like morphology in the aligned fibers, whereas they maintained a mostly flat stellar shape in the random fibers. Gene expression profiling of these cells post seeding showed up-regulation of transforming growth factor β-1 (TGFβ-1) along with other mesenchymal biomarkers, suggesting that these cells undergo epithelial-mesenchymal transitions in response to the polymer scaffold. The results of this study indicate that the topographical cue may play a significant role in tumor progression.

Enrichment and Selective Targeting of Cancer Stem Cells in Colorectal Cancer Cell Lines

Cancer stem cells (CSCs) are the subpopulation of cells within a tumor proposed to be responsible for tumor initiation, relapses, and resistance to chemotherapeutic drugs. Here we optimized sphere culture conditions to isolate and enrich CSCs from colorectal cancer cell line IMCE-Ras. Spheroid cells that developed in culture expressed high levels of putative stem cell markers, and showed stronger anchorage-independent growth abilities and resistance to conventional chemotherapeutic drugs compared with the initial monolayer adherent cells. Xenograft transplantation assays further demonstrated that IMCE-Ras spheroid cells are highly enriched in CSCs. To develop CSC-targeted therapy, we found that the relative percentage of CSC in IMCE-Ras cells was significantly decreased after a short duration exposure to DNA Methylation inhibitor 5-aza-2’-deoxycytidine (5-Aza-dC), indicating that DNA Methylation may be critical for self-renewal and maintenance of CSCs. Indeed, double knockout of DNA methyltransferase 1 (DNMT1) and DNA methyltransferase 3b (DNMT3b) in colon cancer cell line HCT116 resulted in loss of >95% DNA Methylation and complete loss of tumorigenicity both in vitro and in vivo. These data suggest that DNA Methylation is critical for maintenance of the colon CSC population, and a combination of classical chemotherapeutic drugs and DNA Methylation inhibitors may be an effective treatment of colon cancer.

Cancer stem cells and cells of origin in MMTV-Her2/neu-induced mammary tumorigenesis.

Lo et al. purport to identify the mammary tumor-inducing cells in mouse mammary tumor virus (MMTV)-neu transgenic mice to be derived from ‘luminal progenitors’. Despite some new experimental data that are mainly confirmatory, the authors do not discuss the fact that multiple research groups had previously reported the originating cells of MMTV-neu tumors. It is our collective opinion that none of the advances reported by the authors are unique to their experimental approach. Using genetically tagged cells in WAP-Cre/Rosa26-lox-STOP-lox-lacZ triple transgenic mice, Henry et al. reported that lacZ-positive parity-identified mammary epithelial cells (PI-MECs) were the targets for MMTV-neu tumorigenesis. In addition, PI-MECs were subsequently shown to be virtually 100% sorted into the CD49f-positive mammary epithelial fraction. Boulanger et al. showed that PI-MECs were multipotent and sensitive to tumor growth factor beta (TGFb) expression, and Booth et al. showed that MMTV-neu induced mammary cancer cells (marked by lacZ expression) were suppressed by the normal mammary microenvironment but still maintained some of their multipotency. None of these reports were referenced or discussed by Lo et al., although all of them are directly related to the experimental subject of this manuscript. These omissions are egregious and suggest at a minimum that the authors had not previewed the earlier literature relating to their studies. More importantly, the previous reports indicate that the targets for MMTV-neu are not ‘luminal progenitors’ but alveolar progenitors that are multipotent, express CD49f and are inhibited from expansion by both TGFb expression and the absence of cyclin D1. These previously published works seriously diminish the scientific impact of the paper published by Lo et al. Further, in the light of these previous reports, the alleged novelty of the findings described in the article published by Lo et al. is misleading and a note of correction from Oncogene or these authors is appropriate.

Abstract 5347: Identification of differentially expressed proteins in breast cancer stem cells using proteomic profiling

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnCancer stem cells (CSCs) are now recognized as pivotal entity in the development and progression of breast cancer. The CSCs are believed to be responsible for recurrence/relapse of cancer because of their ability to resist chemotherapeutic drugs. The present drugs in the clinics predominantly target the highly proliferating cells sparing the slow dividing and resistant cells. If the drugs targeting the cancer stem cells evolve, there is a possibility of eradication of cancer. In this direction it is imperative to understand the fundamental difference between the cancer stem cells and the non cancer stem cells. Hence, a basic understanding of genes expressed in CSCs vs non-CSCs needs to be explored. A large number of high throughput and extensive Micro array studies have been carried out to understand the normal and various breast cancer subtypes however, the RNA profiles does not necessarily match the protein profile, Therefore determination of protein levels is the key to identify the novel targets for cancer stem cells. To address this issue and to identify meaningful targets for breast cancer we have characterized breast cancer stem cells from primary cultures of Her2/Neu transgenic mice. We identified the altered proteins through LC-MS/MS using an LTQ-orbitrap mass spectrometer and confirmed those results using quantitative RT-PCR. We further performed insilico analysis, using microarray datasets, for identification of group of genes, which in combination can provide prognostic importance. We identified PTMA, S100A4, S100A6, TNXRD1, COX-1, COX-2 and FTH1 as a group of genes, which in combination were significantly associated with poor clinical outcome of breast cancer patients. Among these genes FTH1 was identified as a novel protein associated with prognosis of breast cancer, which is overexpressed in enriched breast CSCs.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5347. doi:1538-7445.AM2012-5347

Abstract LB-203: Mathematical modeling of the dynamic interaction between cancers stem cells and non-stem cancer cells and its potential clinical implication

Cancer stem cells (CSCs) have been identified in primary breast cancer tissues and cell lines. The size of CSC population varies a lot among cancer tissues and cell lines but is associated with aggressiveness of breast cancer. Despite of the high tumor heterogeneity among different samples, CSCs isolated from either primary tissues or cell lines can rapidly regenerate the original heterogeneity and maintain a relatively constant proportion of CSCs. It remains largely unknown how the CSC population is maintained within a tumor. In this study, we develop a mathematical model to explore the key factors which control the size of CSC population during tumor cell growth both in vitro and in vivo. Our mathematic modeling and experimental data suggest that there is a negative feedback mechanism to control the balance between CSC and non-stem cancer cells. We further calculated how feedback sensitivities and robustness can be regulated by different intrinsic and extrinsic factors. As an example, our mathematical model predicts that overexpression of oncogeneHER2 leads to increase in the proportion of CSC via regulating the division mode or proliferation rate of CSCs. The prediction is supported by both in vitro cell culture results and the clinical data. We also show that enrichment of CSCs in suspension culture is due to both depletion of differentiated cancer cells and partial relieve of negative feedback. Mathematical modeling this culture condition reveals that the majority of tumporspheres are derived from progenitor cancer cells but not CSCs. Overall, these findings contribute to our understanding of cancer heterogeneity and may have significant implications in development and selection of anti-cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-203. doi:1538-7445.AM2012-LB-203

Abstract 4519: In vitro cell and in vivo animal studies of the demethylating agent in treating HER2-amplified breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnPURPOSE: The recent evidence shows that HER2-amplified breast cancers exhibit more frequent CpG island methylation than other types of breast cancers, suggesting epigenetic alterations are involved in HER2-mediated tumorigenesis. In this study, we addressed the possibility of the use of the demethylating agent (5-aza-2’-deoxycytidine, decitabine) for treatment of HER2-overexpressing breast cancer.nnEXPERIMENTAL DESIGN: Two cell model systems were used in this study: murine primary Her2-overexpressing tumor cell line (A6O5, derived from the tumor naturally developed in HER2/neu transgenic mice) and human HER2-overexpressing luminal breast cancer cell line MCF7-HER2-18. Cells were cultured in regular cell culture dishes with the serum-containing medium (adhesive culture) or in ultra-low attachment dishes with the serum-free sphere culture medium (tumorsphere culture) for drug testing. Tumors developed from A6O5 cells injected into mammary glands of HER2/neu transgenic mice were used as the xenograft model. The early spontaneous mammary tumors naturally developed in HER2/neu transgenic mice (size < 5 mm) were used for drug treatment.nnRESULTS: The in vitro growth of A6O5 was more significantly inhibited by decitabine than that of non-tumorigenic murine mammary epithelial cells. Consistently, MCF7-HER2-18 cells were more sensitive to decitabine-induced growth suppression than its control vector-transfected cell line MCF7-Neo. These results suggest that HER2 overexpression renders cancer cells more sensitive to the effect of decitabine. Surprisingly, the tumorsphere formation of A6O5 and MCF7-HER2-18 was dramatically abrogated by decitabine, suggesting that self-renewal of cancer stem cells in these two cell lines was annihilated by decitabine. Importantly, decitabine inhibited the in vivo growth of A6O5 xenograft tumors. For proof-in-principle of this study, we treated the HER2/neu transgenic mice bearing early developing mammary tumors with decitabine. Indeed, decitabine effectively suppressed the in vivo tumor growth of the decitabine-treated group compared to that of the vehicle-treated group.nnCONCLUSIONS: This study for the first time explores the potential of the demethylating agent in the treatment of HER2-amplified breast cancers using in vitro cell and in vivo animal models. These findings shed light on the translational application of epigenetic modifiers with the HER2-signaling blockers to treatment of HER2-amplified breast cancer patients.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4519. doi:10.1158/1538-7445.AM2011-4519