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Mizejewski. J Cancer Metastasis Treat 2018;4:27 I http://dx.doi.org/10.20517/2394-4722.2018.20 Page 3 of 7

detected were distinct and none clustered with their corresponding cells and tissues from the primary BC
mass. Some of the biomarker constituents of the CTC mRNA signature included cell activities involving
growth regulation, cell adhesion, cell-to-cell contact, spreading, migration, and motility. Such biomarkers
included CD86, PARP6, ERα, GBP2, Adam-17, DDIT4, SLC2A3, SRGN, and NOTCH-1. Additional
biomarkers were involved with chemotaxis, pro-inflammatory factors and immunomodulatory networks
which included CD44, CD45, CD24, TNF, IL-1B, NFkB, CXCL8, CXCR4, and PDGF-BB. Brain-related
biomarkers encompassed NCAM, Serpin I1, plasmin, neuroserpin B2, and UPAR which are required for
stealth transpassage through the blood brain barrier [Supplementary Table 1]. It is of interest that proteins
[14]
such as plasmin, serpins, and UPAR are especially crucial to CTCs for brain entry .

A circumspect examination of some of the gene constituents of the CTC signature revealed that proteins
related to various cellular activities and pathways could be parsed into several functional sub-groups. These
groups displayed mRNA transcripts that were either enhanced (up-regulated) or reduced (down-regulated)
in the blood circulating cells. The regulated gene transcripts encompassed cell activities such as: (1) growth
and proliferation; (2) DNA transcription and translation; (3) signal transduction; (4) cell invasiveness and
migration; and (5) mitotic and metabolic events. In summation, one could deduce from the above listing that
CTC’s appeared to be groomed for maintaining a metabolic “status quo” semi-dormancy state in order to
survive in the blood circulation while preparing for migration to a distant organ site [4,15] . While so doing, the
CTC have to retain their functional cell maintenance in order to detach from cell-to-cell contacts, adhere
to blood platelets, and migrate to target organs (i.e., bone marrow or brain) with the aid of inflammatory
chemokine molecules.

PROTEIN-ENCRYPTED PEPTIDES, CTCS, AND METASTASIS
The containment of a class of growth factor, extra-cellular matrix, and angiogenic peptide fragments
encrypted within the polypeptide chain of a full-length protein is known but is not widely recognized.
However, some of the most potent growth inhibitors are derived from short peptide fragments (segments)
already existent in naturally-occurring mammalian full length proteins that themselves affect cell growth
and proliferation in an opposite function from the mother proteins. This less-recognized concept of a protein-
derived reserve containing peptide growth Inhibitor fragments is becoming a recurring theme in the field
of growth regulation, intracellular signaling, and cross-talk between signal transduction pathways. Classical
examples of such occult (cryptic) peptides include the following examples; (1) tenacin binding peptide
[18]
[16]
[17]
derived from fibronectin ; (2) angiostatin from plasmin ; (3) endostatin from type XVIII collagen ; (4)
[19]
[20]
vasostatin from calreticulin ; and (5) constatin from type-IV collagen . Such cryptic peptide sites can be
exposed following a conformational change on a protein or can be released following proteolytic cleavage
from a larger protein. These peptides can also be chemically synthesized as single fragments of 20-45 amino
acids. A well-published example of a peptide site revealed following a conformational transition change on a
full-length protein is an encrypted “growth inhibitory” site on alpha-fetoprotein (AFP), normally a growth
promoting molecule [21-23] . The encrypted peptide segment, termed the growth inhibitory peptide (GIP), is a
34 amino acid segment concealed in a hydrophobic cleft of the completely-folded AFP molecule. The GIP site
is revealed following protein unfolding in chemical environments containing high ligand concentrations of
estrogens, fatty acids, and growth factors. This transitory GIP form converts the usually growth-enhancing
AFP molecule into a growth-inhibiting polypeptide. This conversion occurs via protein un-folding into a
conformational change resembling the denatured intermediate state of a molten globular form (MGF) of
[22]
protein . Since the MGF of AFP is a transitory intermediate form, AFP can refold back to its native tertiary
fold following excess ligand removal. Because the AFP-MGF form is unstable, the GIP segment itself has
[23]
now been synthesized, purified, and characterized as a distinct 34-mer synthetic peptide segment . The
34-mer GIP fragment can inhibit both growth factor and estrogen-induced growth in a concentration-
dependent fashion in addition to blocking metastatic-associated activities [24,25] .