Michele L. Dziubinski

The Notch pathway is important in maintaining the cancer stem cell population in pancreatic cancer.

Background Pancreatic cancer stem cells (CSCs) represent a small subpopulation of pancreatic cancer cells that have the capacity to initiate and propagate tumor formation. However, the mechanisms by which pancreatic CSCs are maintained are not well understood or characterized. Methods Expression of Notch receptors, ligands, and Notch signaling target genes was quantitated in the CSC and non-CSC populations from 8 primary human pancreatic xenografts. A gamma secretase inhibitor (GSI) that inhibits the Notch pathway and a shRNA targeting the Notch target gene Hes1 were used to assess the role of the Notch pathway in CSC population maintenance and pancreatic tumor growth. Results Notch pathway components were found to be upregulated in pancreatic CSCs. Inhibition of the Notch pathway using either a gamma secretase inhibitor or Hes1 shRNA in pancreatic cancer cells reduced the percentage of CSCs and tumorsphere formation. Conversely, activation of the Notch pathway with an exogenous Notch peptide ligand increased the percentage of CSCs as well as tumorsphere formation. In vivo treatment of orthotopic pancreatic tumors in NOD/SCID mice with GSI blocked tumor growth and reduced the CSC population. Conclusion The Notch signaling pathway is important in maintaining the pancreatic CSC population and is a potential therapeutic target in pancreatic cancer.

Y-box-binding protein 1 confers EGF independence to human mammary epithelial cells

The epidermal growth factor receptor (EGFR) is linked to poor outcome in breast cancer, and resistance to hormonal therapy is often accompanied by activation of growth factor receptors. To investigate the mechanism(s) by which EGFR becomes activated in breast cancer, we screened a cDNA expression library for genes that mediate EGF-independent proliferation of human mammary epithelial cells (HMECs). We isolated the NSEP1 cDNA encoding Y-box-binding protein 1 (YB-1), a multifunctional transcriptional and translational regulator. This cDNA conferred growth factor independence to HMECs. YB-1-transduced cells overexpressed EGFR, but ErbB-2 (Her-2/neu) levels were unchanged. Moreover, EGFR was constitutively phosphorylated in the absence of exogenous ligand. In these cells, an EGFR-blocking antibody failed to inhibit proliferation, conditioned medium activity could not be detected, and the synthesis of EGFR ligands was reduced compared to parental cells. This suggests that EGFR is activated in a ligand-independent fashion. However, cell growth could be blocked with an ErbB kinase inhibitor, indicating that EGFR signaling plays a major role in YB-1-induced growth factor independence. Taken together, our results demonstrate that YB-1 overexpression can induce EGF independence in HMECs via activation of the EGFR pathway. This could represent one of the mechanisms by which YB-1 contributes to breast tumor aggressiveness.

Comprehensive analysis of oncogenic effects of PIK3CA mutations in human mammary epithelial cells.

More than 20 different PIK3CA gene mutations were identified in breast cancer with different frequencies. Whether these breast cancer associated mutations have similar biological effects is largely unknown. In this study, we established a novel cell model using the lentivirus system to express 10 different PIK3CA genes (wild type and mutant) based on the human mammary epithelial cell MCF10A. We found that nine different PIK3CA mutants harbor different abilities to promote cell proliferation and EGF independent growth. In addition, most PIK3CA mutants (except for the wild type PIK3CA, the Q60K and the K111N mutants) had the ability to change the morphogenesis of the MCF10A cell in 3D Matrigel assay. Moreover, different PIK3CA mutants have different abilities to promote colony formation and cell invasion. We further observed that most of the PIK3CA mutants could activate p-AKT and p-p70-S6K in the absence of EGF stimulation. Finally, LY294002, a PI3K inhibitor, can effectively inhibit cell growth in cell lines with different PIK3CAs. Taken together, our results support the notion that different PIK3CA mutations differentially contribute to breast cancer transformation, and exploration of the therapeutic application of these mutations will benefit breast cancer patients with the PIK3CA mutations.

Insulin‐like growth factor and epidermal growth factor independence in human mammary carcinoma cells with c‐erbB‐2 gene amplification and progressively elevated levels of tyrosine‐phosphorylated p185erbB‐2

Growth factor‐independent proliferation is an essential aspect of the transformation process. To study the influence of c‐erbB‐2 overexpression on the autonomous growth of human mammary cancer cells, we used a series of non‐neoplastic and neoplastic human mammary epithelial cell lines isolated from a patient with intraductal and invasive ductal carcinoma of the breast. The non‐neoplastic cell line, H16N‐2, which expresses a normal level (single gene copy) of c‐erbB‐2, was used for comparison with the neoplastic cell lines. Both the metastatic tumor cell lines, 21MT‐1 and 21MT‐2, showed equivalent amplification of the c‐erbB‐2 gene; however, 21MT‐1 cells showed a higher level of c‐erbB‐2 overexpression. Therefore, the H16N‐2, 21MT‐2, and 21MT‐1 cell series forms a distinct gradient of progressively increasing c‐erbB‐2 gene expression. Furthermore, the overexpression of c‐erbB‐2 in the 21MT cell lines was concordant with increases in the constitutive tyrosine kinase activity of p185erbB‐2 measured in the absence of exogenous growth factors in culture. Normal mammary epithelial cells require both insulin‐like growth factor (IGF)‐1 (or supraphysiological concentrations of insulin) and epidermal growth factor (EGF) to proliferate under serum‐free conditions in culture. By contrast, 21MT‐2 cells showed a reduced requirement for IGF but still required EGF to proliferate. 21MT‐1 cells did not require either insulin or EGF to proliferate. Therefore, the progressive increases in constitutive p185erbB‐2 tyrosine kinase activity in the 21MT‐2 and 21MT‐1 cell lines was directly correlated with IGF independence and combined IGF and EGF independence under defined conditions in culture. Experiments using conditioned media and anti‐IGF‐1 receptor and anti‐EGF receptor neutralizing antibodies showed that the growth‐factor independence of the tumor cells did not involve detectable IGF‐ or EGF‐like autocrine activity expressed by the 21MT cells. Furthermore, neu differentiation factor/heregulin, a ligand that indirectly activates p185erbB‐2 by direct binding to erbB‐3 receptors, potently stimulated the proliferation of the growth factor‐dependent H16N‐2 cells (which expressed cerbB‐2 and c‐erbB‐3 but not c‐erbB‐4) in the absence of both IGF and EGF. Thus, HRG‐induced mitogenesis mimicked the autonomous growth seen in the 21MT cells that have the highest level of constitutive p185erbB‐2 activation. These data support the hypothesis that the constitutive activation of p185erbB‐2 in human mammary carcinoma cells causes growth‐factor independence by directly activating multiple signal‐transduction pathways that substitute for both IGF and EGF during proliferation.

GM-CSF Mediates Mesenchymal–Epithelial Cross-talk in Pancreatic Cancer

UNLABELLED Pancreatic ductal adenocarcinoma (PDA) is characterized by a dense stroma consisting of a prevalence of activated fibroblasts whose functional contributions to pancreatic tumorigenesis remain incompletely understood. In this study, we provide the first identification and characterization of mesenchymal stem cells (MSC) within the human PDA microenvironment, highlighting the heterogeneity of the fibroblast population. Primary patient PDA samples and low-passage human pancreatic cancer-associated fibroblast cultures were found to contain a unique population of cancer-associated MSCs (CA-MSC). CA-MSCs markedly enhanced the growth, invasion, and metastatic potential of PDA cancer cells. CA-MSCs secreted the cytokine GM-CSF that was required for tumor cell proliferation, invasion, and transendothelial migration. Depletion of GM-CSF in CA-MSCs inhibited the ability of these cells to promote tumor cell growth and metastasis. Together, these data identify a population of MSCs within the tumor microenvironment that possesses a unique ability, through GM-CSF signaling, to promote PDA survival and metastasis. SIGNIFICANCE The role of stroma in pancreatic cancer is controversial. Here, we provide the first characterization of MSCs within the human PDA microenvironment and demonstrate that CA-MSCs promote tumorigenesis through the production of GM-CSF. These data identify a novel cytokine pathway that mediates mesenchymal-epithelial cross-talk and is amenable to therapeutic intervention. Cancer Discov; 6(8); 886-99. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 803.

UM-164: A Potent c-Src/p38 Kinase Inhibitor with In Vivo Activity against Triple-Negative Breast Cancer.

Purpose: c-Src has been shown to play a pivotal role in breast cancer progression, metastasis, and angiogenesis. In the clinic, however, the limited efficacy and high toxicity of existing c-Src inhibitors have tempered the enthusiasm for targeting c-Src. We developed a novel c-Src inhibitor (UM-164) that specifically binds the DFG-out inactive conformation of its target kinases. We hypothesized that binding the inactive kinase conformation would lead to improved pharmacologic outcomes by altering the noncatalytic functions of the targeted kinases. Experimental Design: We have analyzed the anti–triple-negative breast cancer (TNBC) activity of UM-164 in a comprehensive manner that includes in vitro cell proliferation, migration, and invasion assays (including a novel patient-derived xenograft cell line, VARI-068), along with in vivo TNBC xenografts. Results: We demonstrate that UM-164 binds the inactive kinase conformation of c-Src. Kinome-wide profiling of UM-164 identified that Src and p38 kinase families were potently inhibited by UM-164. We further demonstrate that dual c-Src/p38 inhibition is superior to mono-inhibition of c-Src or p38 alone. We demonstrate that UM-164 alters the cell localization of c-Src in TNBC cells. In xenograft models of TNBC, UM-164 resulted in a significant decrease of tumor growth compared with controls, with limited in vivo toxicity. Conclusions: In contrast with c-Src kinase inhibitors used in the clinic (1, 2), we demonstrate in vivo efficacy in xenograft models of TNBC. Our results suggest that the dual activity drug UM-164 is a promising lead compound for developing the first targeted therapeutic strategy against TNBC. Clin Cancer Res; 22(20); 5087–96. ©2016 AACR.

Isolation and Culture of Human Breast Cancer Cells from Primary Tumors and Metastases

Over the years, a number of groups have developed culture media and conditions that support the growth of normal human mammary epithelial cells from a variety of lineages, and these cell systems have increased our understanding of human mammary gland biology. In contrast, it remains difficult to isolate and culture human breast cancer cells from primary tumors and metastatic specimens. Our laboratory has now developed 13 human breast cancer cell lines. Some of these cell lines originated from primary tumor specimens, others were derived from chest wall recurrences in patients who had prior surgery, and some of the cell lines were derived from pleural effusion metastases. The cell lines that we have obtained exhibit an array of genetic alterations representative of the genomic diversity of uncultured human breast cancers. In addition, these cells are cultured under well-defined conditions (most cell lines are grown in serum-free media) that allow detained cellular studies to be carried out that complement the genetic analyses of these cell lines. This chapter outlines the culture methods and approaches that we have used to develop this panel of cell lines. Our methods emphasize certain key features that we feel are essential for the isolation and culture of breast cancer cells. These features are all focused on developing culture conditions that allow for the slow emergence of cancer cells in the relative absence of normal cells, which proliferate rapidly under highly-growth-factor-enriched conditions. Thus, selective growth media and methods for enriching cell populations for cancer cells while minimizing the presence of normal epithelial and stromal elements are important for the expansion of breast cancer cells in vitro. It is still not possible to develop cell lines from every breast cancer specimen obtained in the laboratory. Thus, whereas the cell lines we have developed have resulted in improvements in our understanding of breast cancer cell biology, much work remains to be done to understand why some breast cancer cells fail to grow in vitro while growing robustly in the patient. Improving our knowledge of the factors that influence breast cancer cell viability and proliferation will be important for development of novel therapeutic strategies for breast cancer.

Usp9x Promotes Survival in Human Pancreatic Cancer and Its Inhibition Suppresses Pancreatic Ductal Adenocarcinoma In Vivo Tumor Growth

Usp9x has emerged as a potential therapeutic target in some hematologic malignancies and a broad range of solid tumors including brain, breast, and prostate. To examine Usp9x tumorigenicity and consequence of Usp9x inhibition in human pancreatic tumor models, we carried out gain- and loss-of-function studies using established human pancreatic tumor cell lines (PANC1 and MIAPACA2) and four spontaneously immortalized human pancreatic patient-derived tumor (PDX) cell lines. The effect of Usp9x activity inhibition by small molecule deubiquitinase inhibitor G9 was assessed in 2D and 3D culture, and its efficacy was tested in human tumor xenografts. Overexpression of Usp9x increased 3D growth and invasion in PANC1 cells and up-regulated the expression of known Usp9x substrates Mcl-1 and ITCH. Usp9x inhibition by shRNA-knockdown or by G9 treatment reduced 3D colony formation in PANC1 and PDX cell lines, induced rapid apoptosis in MIAPACA2 cells, and associated with reduced Mcl-1 and ITCH protein levels. Although G9 treatment reduced human MIAPACA2 tumor burden in vivo, in mouse pancreatic cancer cell lines established from constitutive (8041) and doxycycline-inducible (4668) KrasG12D/Tp53R172H mouse pancreatic tumors, Usp9x inhibition increased and sustained the 3D colony growth and showed no significant effect on tumor growth in 8041-xenografts. Thus, Usp9x inhibition may be therapeutically active in human PDAC, but this activity was not predicted from studies of genetically engineered mouse pancreatic tumor models.

HNF1A is a novel oncogene that regulates human pancreatic cancer stem cell properties

The biological properties of pancreatic cancer stem cells (PCSCs) remain incompletely defined and the central regulators are unknown. By bioinformatic analysis of a human PCSC-enriched gene signature, we identified the transcription factor HNF1A as a putative central regulator of PCSC function. Levels of HNF1A and its target genes were found to be elevated in PCSCs and tumorspheres, and depletion of HNF1A resulted in growth inhibition, apoptosis, impaired tumorsphere formation, decreased PCSC marker expression, and downregulation of POU5F1/OCT4 expression. Conversely, HNF1A overexpression increased PCSC marker expression and tumorsphere formation in pancreatic cancer cells and drove pancreatic ductal adenocarcinoma (PDA) cell growth. Importantly, depletion of HNF1A in xenografts impaired tumor growth and depleted PCSC marker-positive cells in vivo. Finally, we established an HNF1A-dependent gene signature in PDA cells that significantly correlated with reduced survivability in patients. These findings identify HNF1A as a central transcriptional regulator of PCSC properties and novel oncogene in PDA.

HNF1A is a Novel Oncogene and Central Regulator of Pancreatic Cancer Stem Cells

The biological properties of pancreatic cancer stem cells (PCSCs) remain incompletely defined and the central regulators are unknown. By bioinformatic analysis of a PCSC-enriched gene signature, we identified the transcription factor HNF1A as a putative central regulator of PCSC function. Levels of HNF1A and its target genes were found to be elevated in PCSCs and tumorspheres, and depletion of HNF1A resulted in growth inhibition, apoptosis, impaired tumorsphere formation, PCSC depletion, and downregulation of OCT4 expression. Conversely, HNF1A overexpression increased PCSC numbers and tumorsphere formation in pancreatic cancer cells and drove PDA cell growth. Importantly, depletion of HNF1A in primary tumor xenografts impaired tumor growth and depleted PCSCs in vivo. Finally, we established an HNF1A-dependent gene signature in PDA cells that significantly correlated with reduced survivability in patients. These findings identify HNF1A as a central transcriptional regulator of the PCSC state and novel oncogene in pancreatic ductal adenocarcinoma.