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Page 8 of 20 Smigiel et al. J Cancer Metastasis Treat 2019;5:47 I http://dx.doi.org/10.20517/2394-4722.2019.26

engagement of senescence may be an inherent phenomenon that also occurs during other stress-induced
senescence responses (i.e., oncogenes, replication stress, γ-irradiation) among different cell types [200-202] .
Altogether, these findings suggest that, the mesenchymal/stem-cell program engaged during senescence
may have significant negative consequences if those cells can overcome the signals maintaining senescence,
resulting in cells with greater ability metastasize and survive therapy [Figure 2B]. Thus, we suggest that
therapies that target senescent cells would limit the reservoir of aggressive cells harboring a senescence-
associated stemness responsible for therapy failure and relapse. In the following section we will discuss the
potential of targeting E-M/CSC plasticity within malignant, pre-malignant, and senescent populations.

HALTING METASTASIS: TARGETING PRE- AND POST-MALIGNANT CELL PLASTICITY
Liquid biopsies from patients with early-stage BC receiving neoadjuvant therapy can be used to identify
subjects at high risk of recurrence by quantifying the number of CTC. Furthermore, expression of
mesenchymal markers in the CTC correlates with poor prognosis and response to therapy [138,203-206] . With
the advent of single cell analysis techniques, our understanding of the evolution and diversity of tumor cells
that are responsible for invasion, metastasis, and therapy failure is expanding. For example, single-cell qPCR
has identified mesenchymal/CSC gene expression patterns in early-stage breast cancer micro-metastases .
[137]
In contrast, later-stage metastases (from the same PDX tissue) are more heterogeneous, more proliferative,
express differentiation markers, and display greater similarity to the primary tumors. The findings are
consistent with the idea that mesenchymal/CSC initiate metastatic outgrowth at a secondary site, followed
later by increased proliferation and differentiation. More recent single cell RNA-sequencing (scRNA-seq)
studies have confirmed that EMT in primary tumors proceeds through distinct, hybrid states, ranging
from completely epithelial to completely mesenchymal . These E-M hybrids, which harbor the greatest
[207]
level of plasticity, are more efficient at intravasating, extravasating to the lungs, and forming metastases .
[208]
Underlying this biology, E-M hybrids have distinct chromatin landscapes and transcriptional profiles. In situ
analysis identified increasing vascularization and immune cell infiltration (particularly macrophages) nearest
the E-M hybrids and fully mesenchymal cells . A separate scRNA-seq study determined that, in response
[208]
to chemotherapy, emerging chemo-resistant cells undergo transcriptional changes consistent with EMT. In
most patients, this chemo-resistant transcriptional program was not evident before treatment but acquired
via transcriptional reprogramming following treatment . These studies and others make a strong case that
[209]
epithelial tumor cells can be induced into a drug-tolerant, E-M hybrid cell state by chemotherapy [141,209-214] .

Identifying and targeting the pathways responsible for this chemo-resistant reprogramming would help
improve the efficacy of chemotherapy. In a recent example, SRC kinase inhibition prevented the de novo
generation of chemo-resistant cells . Importantly, this chemo-sensitization was temporally dependent, and
[209]
only effective if SRC inhibition occurred after chemotherapy, when the signaling responsible for generating
the chemo-resistance phenotype had become activated. More recently, Cazet et al. identified cross-
[215]
talk between TNBC models and cancer-associated fibroblasts (CAF), which promoted stemness and drug
resistance in the cancer cells via paracrine Hedgehog (Hh) signaling. A key developmental pathway, Hh
signaling requires receptor mediated binding of Hh to Patched (PTCH), resulting in Smoothened (SMO)-
mediated nuclear translocation of Gli1. Gli1 then acts as a transcription factor, mediating Hh pathway
transcriptional changes . Hh signaling is often reactivated in a subset of BC, and specifically in the context
[216]
of TNBC, a paracrine manner . After targeting paracrine Hh signaling in vivo, via two clinically available
[217]
smoothened inhibitors (SMOi) Vismodegib and Sonidegib, Cazet et al. observed a suppression of cancer
[215]
cell plasticity and increased sensitivity to docetaxel. Most importantly, in a phase I clinical trial, 3 of 12
patients with metastatic TNBC observed clinical benefit from combinatorial therapy of SMOi and docetaxel
(one with complete response), similar to treatment paradigms we suggest above. SMOi have been beneficial
for basal cell carcinomas and medulloblastomas where tumors rely on cell-autonomous hedgehog signaling,
however the work here suggests the ability to target the TME in order to dampen cancer cell plasticity, and