A major barrier to the emergence of distant metastases is the survival of circulating tumor cells (CTCs) within the vasculature. Lethal stressors, including shear forces from blood flow, anoikis arising from cellular detachment, and exposure to natural killer cells, combine to subvert the ability of primary tumor cells to survive and ultimately seed distant lesions. Further attenuation of this rate-limiting process via therapeutic intervention offers a very attractive opportunity for improving cancer patient outcomes, in turn prompting the need for a deeper understanding of the molecular and cellular mechanisms underlying CTC viability. MUC4 is a very large and heavily glycosylated protein expressed at the apical surfaces of the epithelia of a variety of tissues, is involved in cellular growth signaling and adhesiveness, and contributes to the protection and lubrication of cellular linings. Analysis of patient-matched breast tumor specimens has demonstrated that MUC4 protein levels are upregulated in metastatic lesions relative to primary tumor among all breast tumor subtypes, pointing to a possible selective advantage for MUC4 overexpression in metastasis. Analysis of a genetically engineered mouse model of HER2-positive breast cancer has demonstrated that metastatic efficiency is markedly suppressed with Muc4 deletion and Muc4-knockout tumor cells are poorly associated with platelets and white blood cells known to support CTC viability. In this review, we discuss the diverse roles of MUC4 in tumor progression and metastasis and propose that intervening in MUC4 intercellular interactions with binding partners on blood-borne aggregating cells could potentially thwart breast cancer metastatic efficiency.

Background

The mammary glands of pigs share many functional and morphological similarities with the breasts of humans, raising the potential of their utility for research into the mechanisms underlying normal mammary function and breast carcinogenesis. Here we sought to establish a model for the efficient manipulation and transformation of porcine mammary epithelial cells (pMEC) in vitro and tumor growth in vivo.

Methods

We utilized a vector encoding the red florescent protein tdTomato to transduce populations of pMEC from Yorkshire -Hampshire crossbred female pigs in vitro and in vivo. Populations of primary pMEC were then separated by FACS using markers to distinguish epithelial cells (CD140a-) from stromal cells (CD140a+), with or without further enrichment for basal and luminal progenitor cells (CD49f+). These separated pMEC populations were transduced by lentivirus encoding murine polyomavirus T antigens (Tag) and tdTomato and engrafted to orthotopic or ectopic sites in immunodeficient NOD.Cg-Prkdc (scid) Il2rg (tm1Wjl) /SzJ (NSG) mice.

Results

We demonstrated that lentivirus effectively transduces pMEC in vitro and in vivo. We further established that lentivirus can be used for oncogenic-transformation of pMEC ex vivo for generating mammary tumors in vivo. Oncogenic transformation was confirmed in vitro by anchorage-independent growth, increased cell proliferation, and expression of CDKN2A, cyclin A2 and p53 alongside decreased phosphorylation of Rb. Moreover, Tag-transformed CD140a- and CD140a-CD49f + pMECs developed site-specific tumors of differing histopathologies in vivo.

Conclusions

Herein we establish a model for the transduction and oncogenic transformation of pMEC. This is the first report describing a porcine model of mammary epithelial cell tumorigenesis that can be applied to the study of human breast cancers.

Mucin-4 (Muc4) is a large cell surface glycoprotein implicated in the protection and lubrication of epithelial structures. Previous studies suggest that aberrantly expressed Muc4 can influence the adhesiveness, proliferation, viability and invasiveness of cultured tumor cells, as well as the growth rate and metastatic efficiency of xenografted tumors. Although it has been suggested that one of the major mechanisms by which Muc4 potentiates tumor progression is via its engagement of the ErbB2/HER2 receptor tyrosine kinase, other mechanisms exist and remain to be delineated. Moreover, the requirement for endogenous Muc4 for tumor growth progression has not been previously explored in the context of gene ablation. To assess the contribution of endogenous Muc4 to mammary tumor growth properties, we first created a genetically engineered mouse line lacking functional Muc4 (Muc4^ko), and then crossed these animals with the NDL (Neu DeLetion mutant) model of ErbB2-induced mammary tumorigenesis. We observed that Muc4^ko animals are fertile and develop normally, and adult mice exhibit no overt tissue abnormalities. In tumor studies, we observed that although some markers of tumor growth such as vascularity and cyclin D1 expression are suppressed, primary mammary tumors from Muc4^ko/NDL female mice exhibit similar latencies and growth rates as Muc4^wt/NDL animals. However, the presence of lung metastases is markedly suppressed in Muc4^ko/NDL mice. Interestingly, histological analysis of lung lesions from Muc4^ko/NDL mice revealed a reduced association of disseminated cells with platelets and white blood cells. Moreover, isolated cells derived from Muc4^ko/NDL tumors interact with fewer blood cells when injected directly into the vasculature or diluted into blood from wild type mice. We further observed that blood cells more efficiently promote the viability of non-adherent Muc4^wt/NDL cells than Muc4^ko/NDL cells. Together, our observations suggest that Muc4 may facilitate metastasis by promoting the association of circulating tumor cells with blood cells to augment tumor cell survival in circulation.

The lethality of the aggressive brain tumor glioblastoma multiforme (GBM) results in part from its strong propensity to invade surrounding normal brain tissue. Although oncogenic drivers such as epidermal growth factor receptor activation and Phosphatase and Tensin homolog inactivation are thought to promote the motility and invasiveness of GBM cells via phosphatidylinostitol 3-kinase activation, other unexplored mechanisms may also contribute to malignancy. Here we demonstrate that several components of the planar cell polarity (PCP) arm of non-canonical Wnt signaling including VANGL1, VANGL2 and FZD7 are transcriptionally upregulated in glioma and correlate with poorer patient outcome. Knockdown of the core PCP pathway component VANGL1 suppresses the motility of GBM cell lines, pointing to an important mechanistic role for this pathway in glioblastoma malignancy. We further observe that restoration of Nrdp1, a RING finger type E3 ubiquitin ligase whose suppression in GBM also correlates with poor prognosis, reduces GBM cell migration and invasiveness by suppressing PCP signaling. Our observations indicate that Nrdp1 physically interacts with the Vangl1 and Vangl2 proteins to mediate the K63-linked polyubiquitination of the Dishevelled, Egl-10 and Pleckstrin (DEP) domain of the Wnt pathway protein Dishevelled (Dvl). Ubiquitination hinders Dvl binding to phosphatidic acid, an interaction necessary for efficient Dvl recruitment to the plasma membrane upon Wnt stimulation of Fzd receptor and for the propagation of downstream signals. We conclude that the PCP pathway contributes significantly to the motility and hence the invasiveness of GBM cells, and that Nrdp1 acts as a negative regulator of PCP signaling by inhibiting Dvl through a novel polyubiquitination mechanism. We propose that the upregulation of core PCP components, together with the loss of the key negative regulator Nrdp1, act coordinately to promote GBM invasiveness and malignancy.

Anticancer chemotherapeutics often rely on induction of apoptosis in rapidly dividing cells. While these treatment strategies are generally effective in debulking the primary tumor, post-therapeutic recurrence and metastasis are pervasive concerns with potentially devastating consequences. We demonstrate that the amiloride derivative 5-(N,N-hexamethylene) amiloride (HMA) harbors cytotoxic properties particularly attractive for a novel class of therapeutic agent. HMA is potently and specifically cytotoxic toward breast cancer cells, with remarkable selectivity for transformed cells relative to non-transformed or primary cells. Nonetheless, HMA is similarly cytotoxic to breast cancer cells irrespective of their molecular profile, proliferative status, or species of origin, suggesting that it engages a cell death mechanism common to all breast tumor subtypes. We observed that HMA induces a novel form of caspase- and autophagy-independent programmed necrosis relying on the orchestration of mitochondrial and lysosomal pro-death mechanisms, where its cytotoxicity was attenuated with ROS-scavengers or lysosomal cathepsin inhibition. Overall, our findings suggest HMA may efficiently target the heterogeneous populations of cancer cells known to reside within a single breast tumor by induction of a ROS- and lysosome-mediated form of programmed necrosis.

The resistance of cancer cell subpopulations, including cancer stem cell (CSC) populations, to apoptosis-inducing chemotherapeutic agents is a key barrier to improved outcomes for cancer patients. The cationic amphiphilic drug hexamethylene amiloride (HMA) has been previously demonstrated to efficiently kill bulk breast cancer cells independent of tumor subtype or species but acts poorly toward non-transformed cells derived from multiple tissues. Here, we demonstrate that HMA is similarly cytotoxic toward breast CSC-related subpopulations that are resistant to conventional chemotherapeutic agents, but poorly cytotoxic toward normal mammary stem cells. HMA inhibits the sphere-forming capacity of FACS-sorted human and mouse mammary CSC-related cells in vitro, specifically kills tumor but not normal mammary organoids ex vivo, and inhibits metastatic outgrowth in vivo, consistent with CSC suppression. Moreover, HMA inhibits viability and sphere formation by lung, colon, pancreatic, brain, liver, prostate, and bladder tumor cell lines, suggesting that its effects may be applicable to multiple malignancies. Our observations expose a key vulnerability intrinsic to cancer stem cells and point to novel strategies for the exploitation of cationic amphiphilic drugs in cancer treatment.