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Page 4 of 13 Eng et al. J Cancer Metastasis Treat 2019;5:69 I http://dx.doi.org/10.20517/2394-4722.2019.021

recruitment of downstream proteins to the phagophore assembly site. Next, Atg9 and its cycling system
(Atg2, Atg18) are activated to facilitate membrane delivery to the expanding phagophore. This is followed
by the assembly of the PI3K complex consisting of Vps34, Vps15, Vps30/Atg6, Atg14, and Atg38 [Figure 1],
a process responsible for recruiting more proteins that are required for further phagophore expansion.
Subsequently, two ubiquitin-like conjugation systems are activated for phagophore expansion, namely
the Atg12 and the Atg8 conjugation systems. A detailed mechanism in which these conjugation systems
[10]
function has been reported by Yin et al. . The human homologs of Atg8 are categorized into two families
- LC3 and GABARAP. Importantly, LC3 is employed during the phagophore elongation stage, whereas
GABARAP proteins are only activated at a later stage of maturation. Upon induction of phagophore
elongation, LC3 is cleaved at the C-terminal by ATG4B protease to form cytosolic LC3-I. This is followed
[13]
by its subsequent conjugation and recruitment to the membrane to form LC3-II . The level of LC3-II is
often known to correlate with the number of autophagosomes due to its association with autophagosome
[14]
maturation .
Fundamentally, LC3-II acts as an adaptor protein that selectively recruits cargoes to the autophagosome
to be degraded . Following cargo recruitment and closure of the autophagosome, the autophagosome
[14]
[12]
directly fuses with a lysosome to form an autolysosome . The cytosolic cargoes within the autolysosome
are then broken down by lysosomal hydrolases, liberating building blocks for future synthesis of other
macromolecules.

The importance of autophagy as a cellular homeostatic mechanism is underscored by the prevalence of
autophagic defects in a myriad of diseases. Besides being a protein and organelle quality control mechanism
that prevents the accumulation of aggregated and dysfunctional proteins, basal autophagy is also a highly
regulated catabolic process that supports cellular metabolic and biosynthetic programs in response to
nutrient deprivation and other forms of stress. In cancer pathogenesis, however, autophagy is hijacked by
rapidly proliferating cancer cells as they adapt to the perturbations of the cancer microenvironment .
[15]
Autophagy is finely regulated in response to the specific energy demands or stress cues emitted throughout
the different stages of metastasis - local invasion, intravasation, dissemination, extravasation and
colonization at distant sites. Several studies suggested that autophagy is associated with EMT in cancer
[16]
and activation of autophagy promotes metastasis . This is corroborated by the high levels of autophagy
[17]
detected in EMT-activated cancer cells, hence strongly supporting its role in promoting early metastasis .
Additionally, cell detachment from the extracellular matrix typically has been shown to induce anoikis,
a self-defense strategy that eliminates misplaced cells through apoptosis [18,19] . It then becomes crucial
for cancer cells to evade anoikis so as to survive while traversing to distant organs/tissues. Importantly,
several studies have postulated the pro-metastatic role of autophagy in overcoming anoikis [20-22] . Although
the underlying mechanisms remain largely unknown, anoikis resistance conferred by autophagy has been
shown to enhance the survival of cancer cells when they disseminate into the lymphovascular system,
leading to their successful metastasis to distant organs.

Given that autophagy is a key cellular process that confers stress tolerance under adverse conditions, its
activation in cancer cells that intravasate into the blood or lymphatic vessels enables them to withstand
[23]
extreme metabolic stress induced by the changing environment . To compensate for the sudden
change in energy demands of the circulating DTCs, autophagy is activated to retain pools of functional
mitochondria to sustain viability of the DTCs. In addition, autophagy-mediated degradation of proteins
provides a sustainable source of nutrients for the DTCs to fuel ATP production and biosynthesis of other
macromolecules that are essential for growth and survival [23,24] .

Successful metastatic colonization is not only limited to the ability of DTCs to overcome stress that they
are exposed to during circulation. Following extravasation from the blood vessel, the DTCs have been shown