Expression of MHC Class I on breast cancer cells correlates inversely with HER2 expression

Figures & data

Figure 1. Representative immunostaining of HER2, MHC class I and PTEN. (A) HER2 staining (0, 1+, 2+, 3+). (B) MHC class I staining (weak and strong). (C) PTEN staining (0, 1+, 2+, 3+). Original magnification: 200x.

Table 1. Clinical features of the patients (n = 70)

Age (years old)
Mean	57
Range	31–86
Primary tumor^#1
pTis	1
pT1	40
pT2	25
pT3	2
pT4	2
Lymph node metastasis^#1
pNx	2
pN negative	37
pN positive	31
Estrogen receptor
Negative	28
Positive	42
Progesterone receptor
Negative	27
Positive	42
Histological Classification
1. Ductal carcinoma in situ	1
2. Invasive carcinoma
a. Invasive ductal carcinoma
Papillotubular	17
Solid tubular carcinoma	7
Scirrhous carcinoma	33
b. Special types
Mucinous carcinoma	6
Apocrine carcinoma	1
Invasive micropapillary carcinoma	4
3. Unclassified	1
Nuclear grade ^#1
1	29
2	19
3	20

#1 The grade of tumor and stages were defined according to the UICC (TMN) classification.

Figure 2. Representative MHC class I immunostaining of HercepTest 0 (A) and 3+ (B) cases. Serial sections were stained with antibodies specific for HER2 and MHC class I. (A) MHC class I staining was strong in HercepTest 0 case. (B) MHC class I staining was weak in HercepTest 3+ case. Original magnification: 200x.

Table 2. Summarized immunohistochemical staining data (n = 70)

	MHC Class l		PTEN				Ki67		ER		PgR
	Weak	Strong	0	1+	2+	3+	Low	High	-	+	-	+
HercpTest 0, 1+	30	18	11	19	13	5	37	11	14	34	14	34
Hercp Test 2+, 3+	20	2	3	12	7	0	16	6	16	6	13	3
	p = 0.031		no correlation				no correlation		p = 0.013		p = 0.021

Figure 3. HER2 downregulation by siRNA upregulates MHC class I expression on breast cancer cells. MDA-MB-231 cells were transfected with a non-targeting siRNA (siCont) or with a siRNA for the downregulation of HER2 (siHER2). HER2 expression was downregulated in siHER2 transfectants, as assessed by flow cytometry (A) and immunoblotting (B). (C) MHC class I expression was increased in cells transfected with siHER2, as assessed by cytofluometry upon staining with two different antibodies specific for MHC class I molecules, clone W6/32 and clone G46–2.6. Representative results from n = 5 independent experiments are shown.

Table 3. HER2-siRNA effect on MHC class I expression

Cell line	Incubator time (hours)	HER2 (MFI)		MHC Class I (MFI) clone W6/32		MHC Class I (MFI) clone G42–2.6
		siCont	siHER2	siCont	siHER2	siCont	siHER2
BT549	48	23.4	10.3	215.8	235.1	73.5	78.7
	72	24.9	7.9	202.9	232.9	68.8	71.6
SK-BR-3	48	527.7	303.7	25.8	29.3	13.9	15.5
	72	ND	ND	ND	ND	ND	ND

Abbreviations: MFI, mean florescence intensity; siCont, control siRNA; siHER2, HER2 siRNA; ND, not determined.

Figure 4. MAPK inhibiton upregulates MHC class I expression on breast cancer cells. MDA-MB-231, BT549 and SK-BR-3 were treated with the ERK inhibitor PD98059. Phospho-p44^MAPK and phospho-p42^MAPK levels were reduced in a dose-dependent manner, as assessed by immunoblotting. Conversely, MHC class I expression was increased upon exposure to PD98059 in a dose-dependent manner, as assessed by cytofluometry upon staining with two different antibodies specific for MHC class I molecules, clone W6/32 and clone G46–2.6. Non-viable (7-AAD⁺) cells were excluded from the analysis. Representative results from n = 4 independent experiments are shown. MFI, mean fluorescence intensity.