Session 7

Understanding the metastatic cascade to learn how to inhibit tumor progression


Enrique Diaz Cantón


Mechanosensitive hormone signaling promotes mammary progenitor expansion, cancer risk and malignant progression

Valerie Weaver

Tissue stem cell frequency has been implicated in risk to malignancy and tumor aggression. Tissue-specific factors linking stem cell frequency to cancer risk and progression remain ill defined. Using a genetically engineered mammary mouse model of elevated integrin mechanosignaling, and syngeneic orthotopic manipulations and patient-derived breast tumor xenografts implanted within cross-linked collagen we observed that a stiff extracellular matrix (ECM) and high integrin mechanosignaling increased the number of epithelial cells with a basal-like mesenchymal phenotype and promoted breast tumor metastasis. A stiffened ECM and high integrin mechanosignaling also increased mammary progenitor cell frequency to enhance breast tumor initiation. Upon further investigation we determined that a stiff ECM and high integrin- mechanosignaling potentiated progesterone receptor-dependent RANK signaling that expanded breast epithelial stem/progenitor frequency. Consistently, inhibiting RANKL binding reduced breast epithelial stem/progenitor cell number. The stiff breast tissue from women with high mammographic density also had elevated RANK signaling and an expanded pool of stem/progenitor epithelial cells. The data link tissue fibrosis and elevated integrin mechanosignaling to stem/progenitor cell frequency and causally implicate hormone signaling in this phenotype. Thus we conclude that inhibiting RANK signaling would decrease the frequency of stem/progenitor cells in the breast to reduce the elevated lifetime risk to malignancy associated with high mammographic density and the aggressiveness of highly fibrotic breast tumors.

The mechanism of metastasis during breast cancer progression and how to inhibit it

John Condeelis

Multiphoton microscopy of live animals in real time led to the discovery of cause and effect relationships between cells leading to metastasis, relationships otherwise not possible to identify using in vitro models. This has revolutionized our understanding of cancer metastasis. Multiphoton imaging demonstrates that tumor cells migrate with macrophages and move with high persistence to blood vessels under the control of HGF gradients (1) . At the blood vessel these migrating tumor cells interact with TMEM, the intravasation doorway, composed of a three cell complex involving the direct contact between a Mena-Hi tumor cell, endothelial cell and Tie2-Hi/VEGF-Hi macrophage. The TMEM structure itself, as well as the gene expression pattern of tumor cells interacting with TMEM, have been validated as prognostic markers for predicting metastasis in breast cancer patients (2, 3) . These were the first markers of metastasis in clinical use derived from multiphoton intravital imaging. TMEM doorways are the only sites in breast tumors where transendothelial migration of tumor cells and intravasation occur (4) . TMEM doorways are found in both primary and metastatic tumor sites in breast cancer (5) and in primary and metastatic sites of pancreatic ductal adenocarcinoma. Clinical trials of TMEM inhibitors targeting TMEM doorways in both primary and secondary sites (6, 7) , are now underway in breast cancer patients (clinical trials study number NCT02824575). As tumor cells interact with TMEM doorways, tumor cell crowding occurs around TMEM causing Mena INV expression in cancer cells in response to macrophage- induced NOTCH signaling (8) , and other macrophage-dependent tumor cell programming associated with Cancer Stem Cells (CSCs), metastatic seeding and dormancy (9) . Mena INV expression is necessary for transendothelial migration during intravasation at TMEM doorways (10) and assures the efficient dissemination of seeding competent CSCs and hard to detect dormant tumor cells which, after a delay, contribute to metastatic growth.

The impact of disseminated cancer cell dormancy on the paradigm of metastasis

Julio Aguirre-Ghiso

Increasing evidence shows that cancer cells can disseminate from early-evolved primary lesions much earlier than the classical metastasis models predicted. It is thought that a state of early disseminated cancer cell (early DCC) dormancy can precede genetic maturation of DCCs and metastasis initiation. Here we reveal at single cell resolution a previously unrecognized role of mesenchymal- and pluripotency-like programs in coordinating early cancer cell spread and a long-lived dormancy program in early DCCs. Using in vitro and in vivo models of invasion and metastasis, single cell RNA sequencing and human sample analysis, we provide unprecedented insight into how early DCC heterogeneity and plasticity control the timing of reactivation. We identify in early lesions and early DCCs the transcription factor ZFP281 as an inducer of mesenchymal- and primed pluripotency-like programs, which is absent in advanced primary tumors and overt metastasis. ZFP281 not only controls the early spread of cancer cells but also locks early DCCs in a prolonged dormancy state by preventing the acquisition of an epithelial-like proliferative program and consequent metastasis outgrowth. Thus, ZFP281-driven dormancy of early DCCs may be a rate-limiting step in metastatic progression functioning as a first barrier that DCCs must overcome to then undergo genetic maturation.

Drug repurposing of hemostatic compound desmopressin (dDAVP) in triple-negative breast cancer (TNBC): preclinical antitumor activity on 2D/3D cell growth, chemotaxis, tumor progression and metastatic spread

(Selected from posters)

Juan Garona

Desmopressin (dDAVP) is a repurposed hemostatic drug in oncology that acts as a selective agonist for the AVPR2 receptor present in blood microvessels and some tumor cells. Preclinical data show that compound triggers cytostatic mechanisms in malignant cells, impairing angiogenesis and metastatic progression. It is known that triple-negative breast cancer (TNBC) is associated to poor prognosis due to limited response to therapy and metastatic relapse. Considering the unsatisfied clinical needs of TNBC, we evaluated the antitumoral activity of dDAVP on aggressive preclinical models of TNBC, alone or in addition to chemotherapy. dDAVP significantly reduced clonogenic and 3D growth, viability and chemotaxis of AVPR2-expressing TNBC cells. Cytostatic effects of dDAVP were associated to altered actin cytoskeleton dynamics and differential expression of migration, angiogenesis and metastasis-related genes. Synergistic effects were observed after combining dDAVP with taxane or alkylating therapy. In animals bearing TNBC tumors combined therapy resulted in greater inhibition of tumor progression, reduction of skin infiltration and metastatic spread to lungs. In conclusion, agonist activation of AVPR2 using dDAVP represents an achievable and interesting therapeutic approach to modulate TNBC aggressiveness. We propose dDAVP as a coadyuvant agent for treating this disease, not only in combination with chemotherapy but also administered during the perioperative setting.

Hypoxic microenvironment is associated with acquired resistance to HER2+ breast cancer immunotherapies

(Selected from posters)

Virginia Judith Wolos

Trastuzumab and trastuzumab emtansine (T-DM1) immunotherapies are the treatment of choice for HER2+ breast cancer patients. However, de novo or acquired resistance is still the main obstacle in clinical practice. To study the effect of hypoxia in acquired resistance, we used human mammary carcinoma BT-474 (HER2+) and MCF-7 (control) cell lines. As a hypoxia model, we added CoCl2 (100 µM) in cell culture medium. The hypoxic status of the cells was confirmed by a Western blot analysis showing a peak of HIF-1α expression after 6 hours. This result correlated with VEGF induction, as measured by RT-qPCR (p<0.05). It is known that hypoxia has a role in regulating stem cell behaviour. Accordingly, BT-474 cells treated with CoCl2 developed a higher number of mammospheres than normoxic cells (p<0.01). Then, we studied the hypoxia-mediated effect on trastuzumab and T-DM1 cell treatments. We found that BT-474 cells treated with increasing concentrations of the drugs for 72 hours presented a significantly higher viability under the hypoxic condition (p<0.05), showing its cytoprotective effect. In MCF-7 cells, both drugs were less effective and no significant differences between conditions were found. In addition, a flow cytometry analysis showed that the drugs decreased membrane HER2 protein expression (p<0.01) regardless of hypoxic microenvironment. Cellular mechanisms underlying the role of hypoxia in acquired resistance to HER2+ breast cancer immunotherapies are being studied.