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Wallace et al. J Cancer Metastasis Treat 2019;5:9 I http://dx.doi.org/10.20517/2394-4722.2019.01 Page 5 of 18

cancer progression, with over 40% of breast cancers presenting with constitutive STAT3 activation. Because
STAT3 activates multiple signaling pathways, aberrant activation can promote multiple changes associated
with cancer, including altered cell cycle dynamics, EMT, angiogenesis, and interestingly, resistance to cell
death [70,71] . Therapies targeting STAT3 may, thus, be of important therapeutic value for PPBC patients. For
[70]
more about the role of STAT3 in breast cancer, see Segatto et al. .

Removal of the overwhelming number of dead cells from the gland requires the Rac-1 mediated switch of
MECs from a secretory to a phagocytic phenotype - a process which is essential for proper remodeling in
[72]
the second phase of involution . Despite the massive wave of cell death that occurs during the first phase,
if suckling resumes within this window (48-72 h, in mice), involution can be reversed, and lactation can
proceed. This reversibility is due, in large part, to the expression of tissue inhibitors of metalloproteinases
[73]
(TIMPs) . TIMPs preserve the reversibility of gland regression by delaying irreversible tissue remodeling
[74]
events through the inhibition of matrix metalloproteinases (MMPs) . Interestingly, TIMP expression may
also serve as an additional mechanism to regulate epithelial apoptosis, as TIMP3 has been implicated in the
[75]
regulation of cell death in a tumor necrosis factor (TNF)-dependent manner . The number of compensatory
mechanisms that exist to activate apoptosis underscore the importance of postpartum involution to future
rounds of successful lactation. At approximately day three of involution in mice, downregulation of TIMPs
results in activation of the MMPs that degrade the mammary ECM, causing MECs to lose contact with their
[73]
underlying basement membrane . In the absence of pro-survival signaling from the ECM, detached MECs
die by anoikis, thus comprising the second wave of cell death [62,73] . Similar to the first phase, clearance of
dead cells during the second phase of involution is largely mediated by phagocytic MECs, with additional
limited support from professional phagocytes. Milk fat globule epidermal growth factor 8, which works by
recognizing phosphatidylserine on the outer leaflet of the plasma membrane of dying cells, is also critical for
[76]
apoptotic cell clearance during the second phase .

What allows some cells to die and others to live during involution remains largely unanswered. To date,
more than 50 mammary specific knockouts have been generated that exhibit alterations in postpartum
involution - either delayed or premature - that are consistent with the proposed role for each molecule in the
process. For example, activation of pro-survival pathways, such as Akt1, and/or deletion of death inducing
genes, such as Bax, results in delayed involution, while deletion of Akt1 and anti-apoptotic Bcl-x results in
[80]
premature involution [77-80] . See Radisky et al. for an extensive review of these models. As pro-survival
signals progressively decline and pro-apoptotic signals increase during the first phase of involution, the
[81]
mammary basement membrane and ECM become the primary mediators of cell survival . Yet, during
the second phase, the basement membrane and ECM are degraded by proteases. Previously, SEMA7A
mRNA expression was shown to increase in whole mammary extracts during the early phase of involution,
[82]
and its expression was attributed to its immunomodulatory role . Recently, we published that SEMA7A
[57]
is expressed on Epcam+ MECs during the remodeling phase of involution ; however, the downstream
mechanisms activated by epithelial SEMA7A are not well understood. Interestingly, SEMA7A is a ligand for
b1-integrin, which is the receptor for ECM molecules that normally facilitate epithelial cell attachment. It is
therefore possible that SEMA7A may activate b1-integrin signaling and provide a pro-survival mechanism to
overcome anoikis. Furthermore, b1-integrin mediates the activation of known survival pathways including
MAP-K and AKT, which affects survival via stabilization of NF-kB and expression of COX-2. Thus,
SEMA7A may promote anoikis-resistance during involution via activation of b1-integrin in a manner that is
independent of ECM; however, additional investigation is required.

The role of COX-2 during involution has also largely been attributed to its role in the modulation of the
[83]
immune milieu . Yet, COX-2 also contributes to cell survival mechanisms and collagen remodeling via
PGE2, which promotes cell growth and proliferation, modulates collagen expression levels, and upregulates
proteinase expression. Studies investigating the effects of postpartum involution in TNBC progression