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Nagayama. J Cancer Metastasis Treat 2021;7:6 I http://dx.doi.org/10.20517/2394-4722.2020.114 Page 7 of 16

Established cancer
Autophagic activity in thyroid cancer
In established cancers, irrespective of those derived from the thyroid or other organs, it is controversial
whether autophagic activity is elevated or not. In thyroid cancers, autophagic activity is reported lower
in PTCs, especially those with lymph node metastasis, by demonstrating lower LC3-II expression
[52]
levels in WB (NOTE - p62 levels were not studied.) and by lower expression levels of LC-3 protein
in immunofluorescence (NOTE - LC3 puncta, not LC3 expression levels, should be quantified in
[53]
immunofluorescence) , while no difference was found in number of LC3 puncta in immunofluorescence
between benign (including normal, Graves’, goiter, and follicular adenoma tissues) and malignant
[54]
(including FTC, PTC, follicular variant PTC, and ATC) lesions . Lower autophagic activity in thyroid
cancer can be explained by the fact that the oncogene-stimulated RAS-RAF-MEK-ERK and mTOR
signaling pathways inhibit autophagy [55,56] . In general, oncogenic proteins inhibit, but oncosuppressor
[57]
proteins induce autophagy . On the other hand, BRAF-activated long non-coding RNA (BANCR), which
is overexpressed in PTCs, contributes to cell proliferation and activates autophagy as demonstrated by
increased and decreased LC3-II levels by overexpression and knockdown (KD), respectively, of BANCR in
[59]
[58]
WB (NOTE - p62 levels were not studied) . BRAF V600E -induced chronic endoplasmic reticulum stress
and NOXA upregulation are also thought to be reasons for BRAF V600E -mediated autophagy activation in
melanoma . The associations of BRAF V600E and higher expression levels of autophagy-related proteins
[60]
[62]
[61]
(LC3 and p62) and higher expression of BECLIN-1 are noted in PTCs. Up-regulation of LC3-II
expression is also reported to be associated with hypoxia-inducible factor-1α (HIF-1α) and telomerase
[63]
reverse transcriptase (TERT) in PTCs . Of note, higher expression levels of autophagy-related proteins do
not necessarily mean higher autophagic activity.
Finally, CMA is shown increased in PTCs as demonstrated by overexpression of lysosome-associated
[64]
membrane protein type 2A (LAMP2A) (NOTE - the flux of this type of autophagy cannot be examined) .
Autophagy as biological markers for thyroid cancer
Different groups report the distinct autophagy-related gene sets as biological markers for thyroid cancer.
[65]
Zhu et al. reported three genes related to prognosis: inositol 1,4,5-triphosphate receptor type 1 (ITPR1),
chemokine C-C motif ligand 2 (CCL2) and cyclin-dependent kinase inhibitor 2A (CDKN2A). Zhang et al.
[66]
reported two genes related to aggressiveness and prognosis: BECLIN-1 and aplasia Ras homolog member I
(ARH1). A positive correlation between LC3-II levels and urinary iodine concentrations is also reported in
[67]
thyroid cancer patients .

Autophagy-targeted therapy
Regarding the results of autophagy-targeted therapy, reports can be divided into three categories; the
first supporting pro-survival/proliferative effect of autophagy on thyroid cancer cells [Table 1], second
supporting anti-survival/proliferative effects [Table 2], and the third suggesting cell-context-dependent
effects [Table 3].

(1) Studies reporting pro-survival/proliferative effect of autophagy on thyroid cancer
In the first group demonstrating the pro-survival effect of autophagy on thyroid cancer cells [Table 1],
anti-tumor effects of autophagy inhibitors - CQ, 3-MA, and genetic KD of autophagy related proteins - are
[69]
inconsistent; CQ is effective in 8505C, FTC133, and TPC1 cells [63,68] as well as BCPAP cells (concentration
[71]
[72]
[70]
used: 10 μM), but not in BCPAP and FRO cells , KHM-5M and C643 cells , and K-1 cells (10-20 μM).
[67]
Another autophagy inhibitor 3-MA is reported to suppress cell growth in BCPAP cells (5 mM), but
[71]
[73]
[74]
not in KHM-5M and C643 cells , TPC1 cells , SW579 cells , and BCPAP [75,76] (1-3 mM). Genetic
ablation of autophagy by ATG5 KD was also shown to kill 8505C and FTC-133 cells , and by ATG7 KD in
[68]
BCPAP and TPC1 cells [63,69] . It should be noted here that CQ has also been reported to have an autophagy-
independent anti-cancer effect [77-79] .