Alvin T. Lo

HER2 signaling pathway activation and response of breast cancer cells to HER2-targeting agents is dependent strongly on the 3D microenvironment

Development of effective and durable breast cancer treatment strategies requires a mechanistic understanding of the influence of the microenvironment on response. Previous work has shown that cellular signaling pathways and cell morphology are dramatically influenced by three-dimensional (3D) cultures as opposed to traditional two-dimensional (2D) monolayers. Here, we compared 2D and 3D culture models to determine the impact of 3D architecture and extracellular matrix (ECM) on HER2 signaling and on the response of HER2-amplified breast cancer cell lines to the HER2-targeting agents Trastuzumab, Pertuzumab and Lapatinib. We show that the response of the HER2-amplified AU565, SKBR3 and HCC1569 cells to these anti-HER2 agents was highly dependent on whether the cells were cultured in 2D monolayer or 3D laminin-rich ECM gels. Inhibition of β1 integrin, a major cell–ECM receptor subunit, significantly increased the sensitivity of the HER2-amplified breast cancer cell lines to the humanized monoclonal antibodies Trastuzumab and Pertuzumab when grown in a 3D environment. Finally, in the absence of inhibitors, 3D cultures had substantial impact on HER2 downstream signaling and induced a switch between PI3K-AKT- and RAS-MAPK-pathway activation in all cell lines studied, including cells lacking HER2 amplification and overexpression. Our data provide direct evidence that breast cancer cells are able to rapidly adapt to different environments and signaling cues by activating alternative pathways that regulate proliferation and cell survival, events that may play a significant role in the acquisition of resistance to targeted therapies.

Evidence for formation of DNA repair centers and dose-response nonlinearity in human cells

The concept of DNA “repair centers” and the meaning of radiation-induced foci (RIF) in human cells have remained controversial. RIFs are characterized by the local recruitment of DNA damage sensing proteins such as p53 binding protein (53BP1). Here, we provide strong evidence for the existence of repair centers. We used live imaging and mathematical fitting of RIF kinetics to show that RIF induction rate increases with increasing radiation dose, whereas the rate at which RIFs disappear decreases. We show that multiple DNA double-strand breaks (DSBs) 1 to 2 μm apart can rapidly cluster into repair centers. Correcting mathematically for the dose dependence of induction/resolution rates, we observe an absolute RIF yield that is surprisingly much smaller at higher doses: 15 RIF/Gy after 2 Gy exposure compared to approximately 64 RIF/Gy after 0.1 Gy. Cumulative RIF counts from time lapse of 53BP1-GFP in human breast cells confirmed these results. The standard model currently in use applies a linear scale, extrapolating cancer risk from high doses to low doses of ionizing radiation. However, our discovery of DSB clustering over such large distances casts considerable doubts on the general assumption that risk to ionizing radiation is proportional to dose, and instead provides a mechanism that could more accurately address risk dose dependency of ionizing radiation.

Patterned Collagen Fibers Orient Branching Mammary Epithelium through Distinct Signaling Modules

For decades, the work of cell and developmental biologists has demonstrated the striking ability of the mesenchyme and the stroma to instruct epithelial form and function in the mammary gland, but the role of extracellular matrix (ECM) molecules in mammary pattern specification has not been elucidated. Here, we show that stromal collagen I (Col-I) fibers in the mammary fat pad are axially oriented prior to branching morphogenesis. Upon puberty, the branching epithelium orients along these fibers, thereby adopting a similar axial bias. To establish a causal relationship from Col-I fiber to epithelial orientation, we embedded mammary organoids within axially oriented Col-I fiber gels and observed dramatic epithelial co-orientation. Whereas a constitutively active form of Rac1, a molecule implicated in cell motility, prevented a directional epithelial response to Col-I fiber orientation, inhibition of the RhoA/Rho-associated kinase (ROCK) pathway did not. However, time-lapse studies revealed that, within randomly oriented Col-I matrices, the epithelium axially aligns fibers at branch sites via RhoA/ROCK-mediated contractions. Our data provide an explanation for how the stromal ECM encodes architectural cues for branch orientation as well as how the branching epithelium interprets and reinforces these cues through distinct signaling processes.

Constructing Three-Dimensional Models to Study Mammary Gland Branching Morphogenesis and Functional Differentiation

Tissue organogenesis is directed by both intercellular interactions and communication with the surrounding microenvironment. When cells are cultured on two-dimensional plastic substrata (2D), important signals controlling programs of cell proliferation, metabolism, differentiation and death responsible for the formation of correct tissue-specific architecture and function are lost. Designing three-dimensional (3D), physiologically relevant culture models, we can recapitulate some crucial aspects of the dynamic and reciprocal signaling necessary for establishing and maintaining tissue specific morphogenic programs. Here we briefly describe the details of robust methods for culturing mouse primary mammary organoids in 3D gels of different extracellular matrices and describe techniques for analyzing the resulting structures. These designer microenvironments are useful for both understanding branching morphogenesis and signaling integrations, but also for analysis of individual susceptibilities and drug testing.

p53-dependent integration of telomere and growth factor deprivation signals

Ectopically expressed hTERT enables p16INK4A(−) human mammary epithelial cells to proliferate in the absence of growth factors, a finding that has led to the hypothesis that hTERT has growth regulatory properties independent of its role in telomere maintenance. We now show that telomerase can alter the growth properties of cells indirectly through its role in telomere maintenance, without altering growth stimulatory pathways. We find that telomere dysfunction, indicated by 53BP1/phosphorylated histone H2AX foci at chromosome ends, is present in robustly proliferating human mammary epithelial cells long before senescence. These foci correlate with increased levels of active p53. Ectopic expression of hTERT reduces the number of foci and the level of active p53, thereby decreasing sensitivity to growth factor depletion, which independently activates p53. The continuous presence of hTERT is not necessary for this effect, indicating that telomere maintenance, rather than the presence of the enzyme itself, is responsible for the increased ability to proliferate in the absence of growth factors. Our findings provide a previously unrecognized mechanistic explanation for the observation that ectopically expressed hTERT conveys growth advantages to cells, without having to postulate nontelomeric functions for the enzyme.

Epimorphin Is a Novel Regulator of the Progesterone Receptor Isoform-A

Epimorphin/syntaxin-2 is a membrane-tethered protein localized extracellularly (Epim) and intracellularly (Stx-2). The extracellular form Epim stimulates morphogenic processes in a range of tissues, including in murine mammary glands where its overexpression in luminal epithelial cells is sufficient to drive hyperplasia and neoplasia. We analyzed WAP-Epim transgenic mice to gain insight into how Epim promotes malignancy. Ectopic overexpression of Epim during postnatal mammary gland development led to early side-branching onset, precocious bud formation, and increased proliferation of mammary epithelial cells. Conversely, peptide-based inhibition of Epim function reduced side branching. Because increased side branching and hyperplasia occurs similarly in mice upon overexpression of the progesterone receptor isoform-a (Pgr-a), we investigated whether Epim exhibits these phenotypes through Pgr modulation. Epim overexpression indeed led to a steep upregulation of both total Pgr mRNA and Pgr-a protein levels. Notably, the Pgr antagonist RU486 abrogated Epim-induced ductal side branching, mammary epithelial cell proliferation, and bud formation. Evaluation of Epim signaling in a three-dimensional ex vivo culture system showed that its action was dependent on binding to its extracellular receptor, integrin-αV, and on matrix metalloproteinase 3 activity downstream of Pgr-a. These findings elucidate a hitherto unknown transcriptional regulator of Pgr-a, and shed light on how overexpression of Epim leads to malignancy.

Abstract SY02-02: Interaction of MMPs with oncogenic signaling: Disruption of dynamic reciprocity and tissue polarity

Whether or not epithelial cells organize into three-dimensional structures defines their normal or invasive and malignant status. In a model of human breast morphogenesis, we have shown that inhibiting key signaling pathways in breast cancer cells leads to phenotypic reversion of the malignant cells. Using intact architecture or invasive behavior as an endpoint, we report that in all cases, signaling through Raf/MEK/ERK disrupts tissue polarity via MMP9 activity. Induction of Raf or activation of an engineered functionally inducible MMP9 in nonmalignant cells leads to loss of tissue polarity and reinitiates proliferation. Conversely, inhibition of Raf or MMP9 with small molecule inhibitors or shRNAs restores the ability of cancer cells to form polarized quiescent structures. Silencing MMP9 expression reduces tumor growth dramatically in a murine xenograft model. LC-MS/MS analysis comparing secreted proteins from nonmalignant cells – with or without active MMP9 – reveals laminin 111 (LN1) as a prominent target of MMP9. LN1 is shown to be necessary for acinar morphogenesis; thus its degradation by MMP9 provides the molecular mechanism by which tissue polarity is lost and growth and invasion reinitiated. These findings underscore the essential dynamic reciprocity between ECM integrity, tissue polarity, and suppression of Raf/MEK/ERK and MMP9 activities: the correct balance is essential to homeostasis, whereas imbalance leads to malignant progression. Citation 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 SY02-02. doi:10.1158/1538-7445.AM2011-SY02-02