p18INK4C and BRCA1 inhibit follicular cell proliferation and dedifferentiation in thyroid cancer

Figures & data

Figure 1. Loss of p18 stimulates follicular cell proliferation and induces bilateral papillary thyroid tumors. (a) Representative gross appearance and HE staining (inset) of the thyroid tumors from a 9-month-old p18^−/− female mouse. Note the thyroid tumors in both sides of the esophagus (Eso.), Th., Tu., thyroid tumor. (b) Representative HE staining of p18^−/− thyroid tumors. Note typical pathological PTC features including tumor cells containing enlarged, overlapping nuclei (blue arrows), cells with Orphan Annie nuclei (black arrows), and cells with nuclear grooves and nuclear membrane irregularities (yellow arrows). (c) Body weight analysis of p18^−/− mice with or without thyroid tumors. Data represent the mean ± SD of five mice in 2–4 month females without thyroid tumor group, four mice in 2–4 month females with thyroid tumor group, six mice in 4–12 month females without thyroid tumor group, and four mice in 4–12 month females with thyroid tumor group. * p < 0.05 between the group without thyroid tumor and the group with thyroid tumor. (d) IHC analysis of thyroid tumors developed in p18^−/− mice. Calcitonin-positive cells are indicated by arrows. Inset shows the representative IHC analysis of tumor-free thyroid. Note, the majority of tumor cells are negative for calcitonin, other than a few sporadically located calcitonin-positive cells. (e) IHC analysis of tumor-free thyroids from WT and p18^−/− mice at the age of 2–4 months of age. The percentages of Ki67-positive cells were calculated from cells situated in clear duct/gland structures. Results represent the mean ± SD of three animals per group.

Note: * p < 0.05 between the WT group and p18^-/- group.

Figure 2. Heterozygous germline deletion of Brca1 in p18 null mice promotes EMT and enhances metastasis of thyroid tumors. (a) HE staining analysis of thyroid tumors developed in mutant mice. Note relative to p18^−/− thyroid tumor cells, p18^−/−;Brca1^+/- cells are highly heterogenous and poorly differentiated. Tumor a and b show two thyroid tumors derived from two individual p18^−/−;Brca1^+/- mice. Picture in the most right shows lung metastasis from thyroid tumor b. (b, c) IHC analysis of thyroid tumors developed in mutant mice. Note relative to p18^−/− tumor cells, more p18^−/−;Brca1^+/- cells expresses strong VIM and TWIST, but none or weak TTF1, CK19, Galectin-3, and HBME1. (d) IHC analysis of thyroid tumors with an antibody against Ki67. The percentages of Ki67-positive cells were calculated and plotted. Results represent the mean ± SD of three tumors per group.

Note: * p < 0.05 between the p18^-/- group and p18^-/-;Brca1^+/- group.

Table 1 Spontaneous thyroid tumor development in mutant mice ^a

	2-4 months		4-12 months		12-18 months		2-12 months Sub-Total		2-18 months ^b Total
Genotype	F	M	F	M	F	M	F	M	F	M	Total tumor #
WT	0/10	0/9	1/19	0/11	1/16	0/8	1/29	0/20	2/45	0/28	2/73
			(5%)		(6%)		(4%)		(4%)		(3%)
p18^−/−	4/16	0/8	4/15	3/12	4/19 ^c	0/10	8/31 ^d	3/20 ^e	12/50 ^f	3/30 ^g	15/80 ^n
	(25%)		(27%)	(25%)	(21%)		(26%)	(15%)	(24%)	(10%)	(19%)
Brca1^+/-	0/7	0/6	0/8	0/9	0/9	0/7	0/15	0/15	0/24	0/22	0/46
p18^−/−;	5/12	0/5	6/13	6/18	0/14 ^h	5/14	11/25 ^i, k	6/23 ^j, k	11/39 ^l, k	11/37 ^m, k	22/76 ^o, k
Brca1^+/-	(42%)		(46%)	(33%)		(36%)	(44%)	(26%)	(28%)	(30%)	(29%)

^aAll mice were in Balb/c background.

^bAll thyroid tumor bearing mice exhibited hypothyroidism phenotype with significantly small body size, except for a p18^-/-;Brca1^+/- male, a WT female, and a p18^-/- female mice who developed thyroid tumors at the age of 12–18 months.

^cOne of the four mice also developed mammary tumor.

^dP = 0.0265 between WT and p18^-/- females.

^eP = 0.2308 between WT and p18^-/- males.

^fP = 0.0086 between WT and p18^-/- females.

^gP = 0.2377 between WT and p18^-/- males.

^hNo thyroid tumor bearing p18^-/-;Brca1^+/- female mice lived beyond 11 months of age, which was likely caused by hypothyroidism-related developmental and metabolic defects, or by mandatory sacrifice due to bad nutrient condition.

ⁱP = 0.0005 between WT and p18^-/-;Brca1^+/- females.

^jP = 0.0229 between WT and p18^-/-;Brca1^+/- males.

^kNo significance between p18^-/- and p18^-/-;Brca1^+/- groups.

^lP = 0.0049 between WT and p18^-/-;Brca1^+/- females.

^mP = 0.0015 between WT and p18^-/-;Brca1^+/- males.

ⁿP = 0.0016 between WT and p18^-/- mice.

^oP = 0.0001 between WT and p18^-/-;Brca1^+/- mice.

Table 2. Haploid loss of Brca1 in p18-deficient mice activates EMT in thyroid tumor cells.

	Genotype^a
Tumor	Wt	p18^−/−	Brca1^+/-	p18^-/-;Brca1^+/-
Thyroid Tumor	2/73 (3%)	15/80 (19%) ^d	0/46	22/76 (28%)^f
Metastasis^b	0/2	0/15		3/22 (14%)^g
EMT+ tumor No.^c	0/2	3/15 (20%) ^e		16/22 (73%)^h

^aAll mice were in Balb/c background and were at 2–18 months of age.

^bNumber of thyroid tumors metastasized to the lung.

^cAt least an EMT marker (VIM) and two EMT-TFs, which include TWIST, PDGFRβ, p-FRA1, and p-PKCα were detected in > 2% tumor cells by IHC, as we previously reported (Bai, Cancer Res., 2014).

^dA significance from WT and p18^−/− tumors by a two-tailed Fisher’s exact test (p = 0.0016).

^eNo significance from WT and p18^−/− tumors by a two-tailed Fisher’s exact test (p = 1.0000).

^fNo significance from p18^−/−;Brca1^+/- and p18^−/− tumors by a two-tailed Fisher’s exact test (p = 0.1871); but p < 0.0001 from p18^−/−;Brca1^+/- and Brca1^+/- tumors by a two-tailed Fisher’s exact test.

^gNo significance from p18^−/−;Brca1^+/- and p18^−/− tumors by a two-tailed Fisher’s exact test (p = 0.2568).

^hA significance from p18^−/−;Brca1^+/-and p18^−/− tumors by a two-tailed Fisher’s exact test (p = 0.0025).

Figure 3. Overexpression of BRCA1 in human thyroid cancer cells inhibits EMT. 8505C cells transfected with pBabe-empty (empty) and pBabe-HA-BRCA1 (BRCA1) were analyzed by Western blot (a) or qRT-PCR (b). GAPDH was used as a loading control in Western blot.

Note: * p < 0.05 between the empty group and BRCA1 group.

Figure 4. Knockdown of BRCA1 in human thyroid cancer cells promotes EMT and dedifferentiation. B-CPAP (a, b, c) and 8505C (d, e, f) cells infected with pGIPZ-sh-Control (sh-Ctrl) or pGIPZ-sh-BRCA1 targeting different sequences of human BRCA1 (sh-BRCA1-G6 and sh-BRCA1-B7) were analyzed by Western blot (a, d) or qRT-PCR (c, f). * p < 0.05 between the sh-Ctrl group and sh-BRCA1-G6 group, or between the sh-Ctrl group and sh-BRCA1-B7 group. (b, e) The protein levels of each lane in (a) and (d) were quantified, respectively, by Image-Pro Plus 6.0 and normalized by that of GAPDH.

Figure 5. Knockdown of BRCA1 in human thyroid cancer cells promotes EMT and tumorigenesis. (a, b) B-CPAP-sh-Ctrl and B-CPAP-sh-BRCA1-G6 cells were inoculated into the left and right back of four NSG mice, respectively, in a pairwise manner. Similarly, B-CPAP-sh-Ctrl and B-CPAP-sh-BRCA1-B7 cells were inoculated into the left and right back of two NCG mice, respectively, in a pairwise manner. In addition, the same number of B-CPAP-sh-BRCA1-B7 cells were inoculated into the left back of two individual mice. Gross appearance (a) and weight (b) of the tumors formed 5 weeks later were analyzed. Data in (b) represent the mean ± SD of four tumors in each sh-BRCA1 group and six tumors in sh-Ctrl group. * p < 0.05 between the sh-Ctrl group and sh-BRCA1-G6 group, or between the sh-Ctrl group and sh-BRCA1-B7 group. (c, d) Representative tumors derived from sh-Ctrl and sh-BRCA1-B7 cell transplants were analyzed by IHC (c) and Western blot (d). (e) The protein levels of each lane in (d) were quantified by Image-Pro Plus 6.0 and normalized by that of GAPDH (right). Results represent the mean ± SD of four tumors per group.

NOTE: * p < 0.05 between the sh-Ctrl group and sh-BRCA1-B7 group.

Figure 6. Analysis of BRCA1 expression and its correlation with EMT markers in human thyroid tissues and cancers. (a) Analysis of BRCA1 mRNA expression in thyroid cancer samples (Cancer) and paired normal thyroid tissues (Normal) in GEPIA (http://gepia.cancer-pku.cn) dataset. (b) Correlation analysis of mRNA expression of BRCA1 and EMT markers in thyroid carcinoma/NMTC samples (TCGA, Firehose Legacy) by cBioportal for Cancer Genomics (https://www.cbioportal.org).

Data availability statement

All data generated or analyzed during this study are included in this published article and its supplementary information files. All data generated/analyzed during the current study are available from the corresponding auther upon request.