Disrupting Cholesterol-FOXO3-KRT14 axis suppresses metastasis of triple-negative breast cancer

doi:10.21203/rs.3.rs-877843/v1

High metastatic propensity is a devastating challenge to effective therapy of solid tumors, including triple-negative breast cancer (TNBC). In this study, we performed a genome-scale screen and found that cholesterol homeostasis is essential for tumor progression in immunocompetent mice. In an orthotopic TNBC model, intracellular cholesterol promoted lung metastasis by sustaining the expression of keratin 14 (KRT14) and decreasing MHC II, which was indispensable for the cluster metastasis of TNBC. Mechanistic studies demonstrated that FOXO3a regulated the transcription of KRT14. FOXO3a recognized and directly bound to the promoter of KRT14 to inhibit its expression. Cholesterol disrupted the nuclear translocation of FOXO3a by sustaining its phosphorylation, which was regulated by IKK, one of the major upstream kinases of FOXO3a. Apolipoprotein A1 (ApoA1) increased cholesterol efflux and inhibited TNBC metastasis by regulating the IKK-FOXO3a-KRT14 axis. The recombinant adenovirus encoding ApoA1 (ADV-ApoA1) promoted cholesterol efflux, dampened metastasis, and prolonged survival in mice bearing TNBC. Finally, we provided preliminary data showing that high doses of ADV-ApoA1 were well tolerated in rhesus monkeys and Syrian hamsters injected both intravenously and hypodermically. Taken together, cholesterol promotes metastasis via the IKK-FOXO3a-KRT14 axis in TNBC, and facilitating cholesterol efflux from TNBC cells is a promising therapeutic strategy for TNBC clinical treatment.

Cancer Biology

Oncology

Cholesterol

triple negative breast cancer

metastasis

keratin 14

FOXO3a

Apolipoprotein A1

oncolytic virus

Table1 Primers for quantitative real-time PCR (Q-PCR).

Gene	5’foward	3’reverse
Mouse FOXO3a	GAGCTGGAGCTCGAACCTT	TCCTCGGGGATCATGGAGTC
Mouse Ahr	TCTAAGCGACACAGAGACCG	GCAGGGGTGGACTTTAATGC
Mouse p53	GGCGTAAACGCTTCGAGATG	GACTGGCCCTTCTTGGTCTT
Mouse Sp1	GTACAGGACCCCCTTGAGC	GAAAGTTGTGCGGCTGTGAG
Mouse Cebp/α	CTAGGAGATTCCGGTGTGGC	CCCGAGAGGAAGCAGGAATC
Mouse Cebp/β	CGCCTTATAAACCTCCCGCT	TGGCCACTTCCATGGGTCTA
Mouse Klf16	CGTCATGTGCTCCACACTCT	TGGGAATGCCAGTAAACCCC
Mouse CTCF	AGTGAAAATGCTGAGCCGGA	TGTTTGGGCTGGTTGGTTCT
Mouse Gatad2a	CTCGGAGAGACGTGCATCAA	TCTCACCGCTCCTTCTCTCA
Mouse Smad4	GACAAGTTGGCAGCAACAACA	CAAGACATCTGGTGAGCAGGA
Mouse Dpf2	TGCCTGTGACATTTGTGGAAA	GCCATCATCACAGGGGTGAA
Mouse KRT14	CAGAGCGGCAAGAGTGAGAT	GGAAGATGAAAGGTGGGCGT
Mouse ABCA1	CCTCAGCCATGACCTGCCTTGTAG	CCGAGGAAGACGTGGACACCTTC
Mouse CEBP/α	GTGGACAAGAACAGCAACGA	ACGTTGCGTTGTTTGGCTTTA
Mouse CEBP/β	GCTGAGCGACGAGTACAAGA	TTGAACAAGTTCCGCAGGGT
Mouse NFATc1	CCTTTCCGCAACCAGAGGAT	AACCTCCTCTCAGCTCACTCT
Mouse Vimentin	TGAGATCGCCACCTACAGGA	TTGCGCTCCTGAAAAACTGC
Mouse Slug	CATCCTTGGGGCGTGTAAGT	ATGGCATGGGGGTCTGAAAG
Mouse Zeb1	TCCGGCTCCTACTCTCAACA	GCTCCAACCTCCACTGTACC
Mouse SNAIL	ATGGAGTGCCTTTGTACCCG	CAGTAACCACCCTGCTGAGG
Mouse β-actin	GTACCACCATGTACCCAGGC	AACGCAGCTCAGTAACAGTCC
Mouse GAPDH	TCTCCTGCGACTTCAACA	TGTAGCCGTATT- CATTGTCA
E1A	CCGAAGATCCCAACGAGGAG	TTGGCATAGAAACCGGACCC
Human ABCA1	GTTAGGAAACCTGCTGCCCT	ATGCCACACACAGGACGTAG
Human β-actin	TGGCACCCAGCACAATGAAG	CTGTCACCTTCACCGTTCCA

Table2 siRNA for gene knock-down

Gene	Sense strand（5’-3’）	Antisense strand（5’-3’）
FOXO3a-1	GCUCUUGGUGGAUCAUCAATT	UUGAUGAUCCACCAAGAGCTT
FOXO3a-2	GCCAGUCUAUGCAAACCCUTT	AGGGUUUGCAUAGACUGGCTT
Ahr-1	GCAACUAGAGCAACAGCAATT	UUGCUGUUGCUCUAGUUGCTT
Ahr-2	GCAAAUCAACGGCACGUUUTT	AAACGUGCCGUUGAUUUGCTT
p53-1	GACCUAUCCUUACCAUCAUTT	AUGAUGGUAAGGAUAGGUCTT
p53-2	CCACUUGAUGGAGAGUAUUTT	AAUACUCUCCAUCAAGUGGTT
Sp1-1	CCAGCAACAUGGGAAUUAUTT	AUAAUUCCCAUGGUUGCUGGTT
Sp1-2	GCGGCAAAGUAUAUGGCAATT	UUGCCAUAUACUUUGCCGCTT
Cebpa-1	GCGCAAGAGCCGAGAUAAATT	UUUAUCUCGGCUCUUGCGCTT
Cebpa-2	GGAGUUGACCAGUGACAAUTT	AUUGUCACUGGUCAACUCCTT
Cebpb-1	AGGCCAAGAUGCGCAACCUTT	AGGUUGCGCAUCUUGGCCUTT
Cebpb-2	GGAACUUGUUCAAGCAGCUTT	AGCUGCUUGAACAAGUUCCTT
Klf16-1	UGGCGUGUGUGGAUUACUUTT	AAGUAAUCCACACACGCCATT
Klf16-2	GCGCCAAAGCCUAUUACAATT	UUGUAAUAGGCUUUGGCGCTT
CTCF-1	CCUUGCAGGUUGUAAAUAUTT	AUAUUUACAACCUGCAAGGTT
CTCF-2	CCACUUGCGAAAGCAGCAUTT	AUGCUGCUUUCGCAAGUGGTT
Gatada-1	GCACUUCACACAGUAACAUTT	AUGUCACUGUGUGAAGUGCTT
Gatada-2	GCUGCCAAUAAUGAGUUUATT	UAAACUCAUUAUUGGCAGCTT
Smad4-1	GCAGCAGAAUGGAUUUACUTT	AGUAAAUCCAUUCUGCUGCTT
Smad4-2	GCGACGCUGUUCAUAAGAUTT	AUCUUAUGAACAGCGUCGCTT
Dpf2-1	GGAGUAGCCCAGAGCAAUUTT	AAUUGCUCUGGGCUACUCCTT
Dpf2-2	GCGAGUUUCCUGUUAGCAATT	UUGCUAACAGGAAACUCGCTT

Table 3 Serum biochemistry profiles of Syrian hamsters after ADV-ApoA1 injecti

Dosage (VP/Kg)	Sex	ALT (U/L)	AST (U/L)	ALP (U/L)	CK (U/L)	LDH (U/L)	AMY (U/L)	LIP (U/L)	BUN (mmol/L)	CREA (mmol/L)	GLU (mmol/L)
Viehicle	male (n=5)	72.2 ± 18.0	37.2 ± 4.8	189.8±8.2	818.4 ± 301.5	145.6 ± 21.0	1771.7 ± 242.7	54.9 ± 5.1	6.4 ± 1.1	16.2 ± 3.4	12.5 ± 1.7
5x10¹² (DRF-I.H.)	male (n=5)	43.8 ± 9.7	34.6 ± 4.3	232.0 ± 18.4	545.6 ± 239.7	142.0 ± 26.9	2277.3 ± 260.4	37.0 ± 8.2	5.7 ± 0.9	14.0 ± 1.0	9.3 ± 1.0
1.2x10¹² (DRF-I.V.)	male (n=5)	49.6 ± 8.0	51.6 ± 13.2	199.2 ± 20.1	789.4 ± 392.3	193.0 ± 46.7	1840.4 ± 144.1	50.7 ± 7.6	5.3 ± 0.5	15.2 ± 0.8	10.2 ± 2.3
Viehicle	female (n=5)	52.8 ± 7.1	40.8 ± 4.5	201.2 ± 18.9	1005.4 ± 503.0	131.8 ± 31.1	2487.7 ± 405.5	54.5 ± 9.4	7.0 ± 0.8	18.8 ± 2.6	10.3 ± 1.7
5x10¹²(DRF-I.H.)	female (n=5)	35.4 ± 4.8	43.4 ± 4.8	245.2 ± 22.4	871.2 ± 458.9	149.6 ± 30.3	2551.1 ± 199.9	45.0 ± 7.0	5.2 ± 0.8	14.2 ± 3.6	8.9 ± 2.0
1.2x10¹² (DRF-I.V.)	female (n=5)	65.6 ± 23.7	59.4 ± 11.3	201.4 ± 14.9	1153.4 ± 395.6	200.2 ± 47.5	2375.0 ± 517.0	45.7 ± 6.2	6.4 ± 0.5	16.2 ± 2.8	8.8 ± 2.5

I.H., hypodermic injection; I.V., intravenous injection. DRF, dose range finding (12-Day course of 10 viral injections). VP/ PFU =31.5:1. ALT, alanine amiotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; CK, creatine kinase; LDH, lactate dehydrogenase; AMY, serum amylase; LIP, lipase; BUN, blood urea nitrogen; CREA, creatinine; GLU, glucose.

Table 4 Serum biochemistry profiles of adult rhesus monkeys after ADV-ApoA1 injection.

Dosage (VP/Kg)	Sex	ALT (U/L)	AST (U/L)	ALP (U/L)	CK (U/L)	LDH (U/L)	AMY (U/L)	LIP (U/L)	BUN (mmol/L)	CREA (mmol/L)	GLU (mmol/L)
Prior to viral injections	male (n=6), female (n=6)	33.6 ± 13.6	50.1 ± 20.1	647.2 ± 214.7	1036.7 ± 1776.3	597.9 ± 257.9	610.5 ± 145.0	27.4 ± 22.0	7.3 ± 1.7	46.5 ± 6.9	4.9 ± 0.6
5x10¹² (DRF-I.H.)	male (n=1), female (n=1)	31.0 ± 23.0	50.0 ± 5.0	424.0 ± 81.0	1267.0 ± 1112.0	689.0 ± 54.0	689.2 ± 97.2	54.0 ± 38.4	6.9 ± 1.2	46.0 ± 3.0	4.7 ± 0.4
2.4x10¹¹ (DRF-I.V.)	male (n=1), female (n=1)	19.0 ± 5.0	33.5 ± 0.5	803.0 ± 60.0	219.0 ± 96.0	541.5 ± 33.5	615.0 ± 111.9	16.5 ± 3.7	6.3 ± 2.0	44.0 ± 5.0	4.6 ± 0.3
1x10¹³ (MTD-I.H.)	male (n=1), female (n=1)	32.0 ± 2.0	41.5 ± 5.5	575.0 ± 14.0	452.0 ± 175.0	512.0 ± 201.0	419.7 ± 22.0	18.1 ± 3.8	6.3 ± 0.1	47.5 ± 7.5	4.1 ± 0.2
1x10¹³ (MTD-I.V.）	male (n=1), female (n=1)	125.0 ± 16.0 *	70.0 ± 26.0	468.5 ± 5.5	244.5 ± 104.5	571.5 ± 180.5	532.6 ± 44.9	30.2 ± 3.7	7.6 ± 1.4	51.0 ± 4.0	4.8 ± 0.2

I.H., hypodermic injection; I.V., intravenous injection. DRF, dose range finding (12-Day course of 10 viral injections); MTD, maximum tolerated dose (1 viral injection). * p < 0.05, compared with that in untreated group. VP/ PFU =31.5:1. ALT, alanine amiotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; CK, creatine kinase; LDH, lactate dehydrogenase; AMY, serum amylase; LIP, lipase; BUN, blood urea nitrogen; CREA, creatinine; GLU, glucose.

There is NO Competing Interest.

Disrupting Cholesterol-FOXO3-KRT14 axis suppresses metastasis of triple-negative breast cancer

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