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Day 2: August 7, 2018

Metastasis Session

To open this session Dr. Zena Werb from the University of California at San Francisco delivered a presentation on breast stem cells during breast cancer progression and metastasis. Studying breast cancer can be difficult because even in the normal breast there are many different epithelial cell types. Single-cell suspensions from normal breast tissue can be separated into distinct subpopulations such as luminal, basal, and stem cells. Additional markers show that these cells also possess significant heterogeneity even within those subpopulations. Using single-cell mRNA sequencing and unbiased clustering analysis, three distinct epithelial cell populations, one basal and two luminal cell types, were identified.

Heterogeneity of breast cancer cells

In the initial invasive phase of both mouse and human breast cancer, multiple markers show that the leader cells have a different phenotype than the follower cells. Next, human tumor cells from patient-derived mouse xenografts were isolated and analyzed. The low- burden metastatic cells were found to represent either an early or indolent stage in the metastatic process, while the high-burden metastatic cells were from a more advanced stage of the disease. Another difference is that, despite significant heterogeneity, the high-burden cells tend to show a luminal-like phenotype, like the primary tumor, while the low-burden cells tend more toward a basal- or stem-cell–like phenotype. Moreover, the low-burden cells were seen to be highly expressing pluripotency genes and tend to look very different from the primary tumor.

Seeking a means of preventing metastases

Since triple-negative breast cancer cells overexpress MYC, it was hypothesized that inhibition of MYC with dinaciclib might prevent metastasis in mouse xenografts. These studies showed that while the primary tumor growth was slowed, both micro metastases and macro metastases were prevented, but the reasons are not yet fully understood.

Different breast cancer subtypes appear to arise from lineage-specific cell types

Analysis of the single-cell gene signatures suggest there are defined differentiation trajectories for maintenance of homeostasis in the adult human breast. It also appears that there are distinct subpopulations of both basal and luminal lineages that may serve as cells of origin for the different subtypes of breast cancer. Finally, it was observed that there are metastasis-specific and organ-specific differences in cancer; for example, brain metastases are different from bone metastases.

Other insights into the metastatic process in this session included the use of breast cancer stem cells, identified by CD44 and CD24 markers, to study homophilic interactions and metastasis. A bit of humor was added when an “apology” of sorts was offered regarding the name mesenchymal stem cellwhen they are not stem cells in vivo, but take on that character once placed in a petri dish. Names aside, in vivo these cells were found to regulate the extravasation of melanoma cancer cells to the bone marrow and liver. The combination of a cytotoxic agent and an LRP6 blocking antibody may provide therapeutic benefits in patients with breast cancer expressing higher copy numbers of LRP6.

Epigenetics and State Transitions Session

Sequencing has shown that the genome of a treatment-naïve but treatment-responsive tumor is identical to that of the post-treatment, relapsed tumor that is now treatment- resistant. This was believed to occur because of changes in histone methylation patterns leading to altered gene expression. Mouse studies have shown that knocking out a specific histone methyltransferase spontaneously induces medulloblastoma by abolishing the activation of tumor suppressor genes and repression of oncogenes. This observation lead to additional studies showing that drugs that target the KL1 and KL2 domains to control the rate of transcriptional elongation might be used for cancer therapy.

Facilitates chromatin transcription (FACT) is a heterodimeric protein complex and a histone chaperone that is important for transcription. It is especially important in early stem cells, early development, and in tumor-initiating cells. It is less important in normal stem cells in adults. CBL0137, an inhibitor of FACT, is currently being investigated as a potential therapeutic agent in cancer. It interchelates with DNA and changes the structure of chromatin, but does not cause any DNA strand breaks. Soon after treating cells with CBL0137, FACT is immobilized. Normally FACT is mobile and must be to function, but in the presence of CBL0137, it effectively becomes “stuck onto” DNA due to the distortion in the DNA structure. FACT expression is elevated in glioblastoma stem cells compared with matched non-stem tumor cells; in vitro studies have shown that glioblastoma stem cells are extremely sensitive to CBL0137 monotherapy. In combination with cisplatin, CBL0137 greatly reduced the growth of a patient-derived xenograft in mice and the growth of a syngeneic mouse SCLC tumor.

Tumor Microenvironment Session

Vasorin is commonly overexpressed in high-grade gliomas like glioblastomas, and promotes tumor growth and reduces survival in mouse models of glioblastoma. Its expression correlates with increased aggressiveness of human gliomas, suggesting that vasorin may be a potential therapeutic target.

The blood-tumor barrier (BTB) is a major obstacle for drug delivery to brain tumors, which requires crossing the BTB while maintaining the integrity of the normal blood- brain barrier (BBB). Pericyte coverage of tumor vasculature correlates inversely with survival of glioblastoma patients after chemotherapy. Ibrutinib, which selectively targets neoplastic pericytes, disrupts the BTB but not the BBB. Elimination of glioma stem cell- derived pericytes in xenograft models disrupted the BTB tight junctions and increased vascular permeability. Importantly, disruption of the BTB through targeting the tumor pericytes specifically increased drug delivery into glioblastoma and significantly improved efficacy of chemotherapy. These findings highlight the clinical potential of targeting neoplastic pericytes to improve treatment of brain tumors.

A membrane-bound protein, CD55 is a component of the complement regulatory system and also regulates self-renewal and core pluripotency genes. Overexpression of CD55 induces stemness and cisplatin resistance. Kinase-inhibitor treatment of endometrioid cancer cell lines sensitized the CSC to cisplatin and overcame the CD55-induced chemoresistance while not altering the self-renewing character of these cells.

Oncostatin M (OSM), a member of the IL-6 family, is an important driver of mesenchymal and CSC phenotypes. Studies of cell lines from pancreatic ductal adenocarcinomas show that OSM increases stemness, increases migratory behavior, and demonstrate greater therapeutic resistance and tumorigenic potential. OSM expression is high in breast cancer, and therapeutic targeting of OSM in the tumor microenvironment in patients with pancreatic carcinomas may prevent or reverse the aggressive mesenchymal and CSC phenotypes associated with poor outcomes.