The authors have declared that no competing interests exist.
Human uterine leiomyosarcoma (LMS) is neoplastic malignancy that typically arises in tissues of mesenchymal origin. The identification of novel molecular mechanism leading to human uterine LMS formation and the establishment of new therapies has been hampered by several critical points. We earlier reported that mice with a homozygous deficiency for proteasome
Uterine mesenchymal tumours have been traditionally divided into benign tumour leiomyomas (LMA) and malignant tumour leiomyosarcomas (LMS) based on cytological atypia, mitotic activity and other criteria. Uterine LMS, which are some of the most common neoplasms of the female genital tract, are relatively rare uterine mesenchymal tumour, having an estimated annual incidence of 0.64 per 100,000 women
As uterine LMS is resistant to chemotherapy and radiotherapy, and thus surgical intervention is virtually the only means of treatment for this disease
adjuvant therapy is expected to improve the prognosis of the disease. A trend towards prolonged disease-free survival is seen in patients with matrix metalloproteinase (MMP)-2-negative tumours
The mice with a targeted disruption of proteasome
The effects of IFN-γ on expression of PSMB9/b1i was examined using five cell lines
Expression of ER, PR, Ki-67, p53, PSMB9, Calponin hi and Senescence staining in human uterine leiomyosarcoma | ||||||||||||||||||
patient Age in No. yrs | TNN stage | MF | CCN | Immunohi stochemi cal | staining TP53 PSMB9 | CAL. | Somatic mutation | Follow up (■..the) | Senescence | |||||||||
ER PR Ki-67 | JAK1 JAK2 STAT1 PSMB9pro. p53 | p-gal. | PML | |||||||||||||||
1 | 37 | T4N1M0 | 97 | + | - | - | 3000 | +++ | - | - | ND | ND | ND | ND | SM | D(1) | - | - |
2 | 58 | T3N0M3 | 24 | + | - | - | 3500 | * | +/- | - | ND | ND | ND | SM | SM | D(23) | +/- | +/- |
3 | 45 | T2N0MEI | 32 | + | +1- | +/- | 2150 | +++ | - | - | SM | ND | SM | SM | SM | D(24) | - | - |
4 | 65 | T1N0M3 | 30 | + | +/- | +/- | 1700 | +++ | - | - | SM | ND | ND | ND | SM | D(20) | - | - |
5 | 52 | TINEIMa | 107 | + | - | + | 2600 | ++ | + | - | SM | ND | ND | ND | ND | D(13) | + | +/- |
6 | 49 | T1N0M0 | 46 | + | - | - | 4300 | + | - | - | ND | ND | ND | ND | ND | D(24) | - | - |
7 | 55 | T1N0M3 | 75 | + | - | - | 4000 | +++ | - | - | ND | ND | SM | SM | ND | D(!8) | - | - |
8 | 43 | T3N0M0 | 57 | + | + | - | 2000 | - | +/- | +/- | ND | ND | ND | ND | ND | D(10) | +/- | +/- |
9 | 67 | T1N0M0 | 13 | + | - | +1- | 1430 | - | - | - | SM | ND | ND | ND | ND | A(34) | - | - |
10 | 67 | T1N0M0 | 37 | + | - | - | 2100 | - | - | - | ND | ND | SM | SM | ND | A(15) | - | - |
11 | 51 | T1N0M0 | 93 | + | - | - | 4500 | - | - | - | ND | ND | SM | ND | ND | A(94) | - | - |
12 | 48 | T1N0M0 | 14 | + | - | - | 900 | ++ | + | 1- | ND | ND | ND | ND | ND | A(58) | +/- | +/- |
13 | 51 | TIMM@ | 22 | + | +/- | + | 450 | + | - | - | SM | ND | ND | SM | ND | A(34) | - | - |
14 | 67 | T1N0M0 | 64 | + | - | + | 1450 | ++ | - | - | ND | ND | ND | ND | ND | A(15) | - | - |
15 | 52 | T1N0M0 | 65 | + | - | - | 1780 | ++ | - | - | SM | ND | SM | ND | ND | D(23) | - | - |
16 | 42 | T3N0M0 | 73 | + | - | - | 2130 | ++ | - | - | ND | ND | ND | SM | ND | A(21) | - | - |
17 | 80 | T1N0M0 | 98 | + | - | - | 1980 | +++ | - | - | SM | ND | ND | ND | ND | D(19) | - | - |
18 | 56 | T1N0M0 | 78 | + | - | - | 1860 | ++ | - | - | ND | ND | ND | ND | ND | A(11) | - | - |
19 | 58 | T1N0M0 | 40 | + | - | - | 1750 | ++ | - | - | ND | ND | ND | ND | ND | A(10) | - | - |
20 | 65 | T2N0M0 | 67 | + | - | - | 780 | +++ | - | - | SM | ND | ND | SM | ND | A(12) | - | - |
21 | 45 | T1N0M0 | 52 | + | - | - | 1045 | ++ | - | - | SM | ND | ND | SM | ND | A(13) | - | - |
22 | 57 | T2N0M0 | 62 | + | - | - | 980 | ++ | +/- | - | SM | ND | ND | SM | ND | A(11) | +/- | +/- |
23 | 54 | UNDO | 54 | + | - | - | 860 | +++ | - | - | ND | ND | ND | SM | ND | A(02) | - | - |
IFN-γ treatment markedly increased the expression of PSMB9/b1i, a subunit of the immunoproteasome, which alters the proteolytic specificity of proteasomes. After binding of IFN-γ to the type II IFN receptor, which is constructed by two components, IFN-γ receptor subunit 1 (IFNGR1) and IFN-γ receptor subunit 2 (IFNGR2), Janus-activated kinase 1 (JAK1) and JAK2 are activated and phosphorylate the signal transducer and activator of transcription 1(STAT1) on the tyrosine residue at position 701 (Tyr701) and the serine residue at position 727 (Ser727)
The defect was localized to JAK1 activation, which acts upstream in the IFN-γ signal pathway since IFN-γ treatment could not strongly induce JAK1 kinase activity in human uterine LMS cell lines. Sequence analysis demonstrated that the loss of IFN-γ responsiveness in the human uterine LMS cell line was attributable to the inadequate kinase activity of JAK1 due to a G781E somatic mutation in the ATP-binding region
Most frequently, LMS have appeared in the uterus, retroperitoneum or extremities, and although histologically indistinguishable, they have different clinical courses and chemotherapeutic responses. The molecular basis for these differences remains unclear. Therefore, the examination of human uterine LMS tissues
Hayashi ct al. Table 1 | |||||
Mutations in JAK1 kinase, PSMB9 promoter region, and STAT1 in human uterine leiomyosarcoma | |||||
Patient # | JAK1 kinase | PSMB9 promoter region | STAT1(701Y,727S)5 | JAK2 kinase | PSMB9? |
#1 | wt | wt | wt | wt | Neg |
#2 | wt | A210G, C214T(IRF-E)3 | wt | wt | P.Posi |
#3 | Q986P(active) R995S(active)1 | C214T, G219A(IRF-E) | (S710A) 6 | wt | Neg |
#4 | G876R(ATP)2 | wt | wt | wt | Neg |
#5 | C881 F (ATP) | wt | wt | wt | P.posi |
#6 | wt | wt | wt | wt | Neg |
#7 | wt | A216G(IRF-E) | (L693R) 6 | wt | Neg |
#8 | wt | wt | wt | wt | F.pos |
#9 | Y987S(active) | wt | wt | wt | Neg |
#10 | wt | A217G(IRF-E) | (R716S) 6 | wt | Neg |
#11 | wt | wt | (1702L) 6 | wt | Neg |
#12 | wt | wt | wt | wt | F.Posi. |
#13 | Y987S(active) | A216G(IRF-E) | wt | wt | Neg |
#14 | wt | wt | wt | wt | Neg |
#15 | G871E(ATP) | wt | (1702L) 6 | wt | Neg |
#16 | wt | G239A(HSF)'1 | wt | wt | Neg |
#17 | C881 F(ATP) | wt | wt | wt | Neg |
#18 | wt | wt | wt | wt | Neg |
#19 | wt | wt | wt | wt | F.Posi. |
#20 | G873D(ATP) | A210G(IRF-E) | wt | wt | Neg |
#21 | C881Stop(TGC-TGA) | G209T(IRF-E) | wt | wt | Neg |
#22 | Q986P(active) | G215A(IRF-E) | wt | wt | F.Posi. |
#23 | wt | C213A(IRF-E) | wt | wt | Neg |
Hayashi et al. Table 2 | ||||||||
Mutations in the IFN-y pathway in human uterine Ieiomyosarcoma | ||||||||
Gene Name | Locus | GenBank Accession | MIM ID | Tumor | Nucleotide | Amino Acid Domain | Evolutionary4 conservation | |
JAK1 | HUMPTKJAK1 | M64174.1 | 147795 | ULMS | G2612A G2618A | G781E ATP binding G873D ATP binding | p,c,m,r,g,d | |
G2626A | G876R ATP binding | |||||||
G2642T | C881F ATP binding | |||||||
G2643A A2957 C | C881 Stop ATP binding Q986P active site | |||||||
A2960 C | Y987S active site | |||||||
A2985T | R995S active site | |||||||
JAK2 | AF005216 | AF005216.1 | +147796 | ULMS | ND2 | ND ND | p,c,b,m,r,g,d | |
STAT1 | NM_007315 | NM_007315 | +600555 | ULMS | A2104C | 1702L NA3 | c,b,m,r,g,d | |
T2128G | S710A NA | |||||||
T2078 G | L693R NA | |||||||
A2148C | R716S NA | |||||||
PSMB91 | X62741 | X62741.1 | 177045 | ULMS | A209T | IRF-E site | p,c,b,m,r,d | |
A210G | IRF-E site | |||||||
C213A | IRF-E site | |||||||
C214T | IRF-E site | |||||||
G215A | IRF-E site | |||||||
A216G | IRF-E site | |||||||
A217G | IRF-E site | |||||||
G219 A | IRF-E site | |||||||
G239 A | HSF site |
In a recent report, a comparative genomic hybridization (CGH)-based analysis of human LMS using a high resolution genome-wide array gave gene-level information about the amplified and deleted regions that may play a role in the development and progression of human uterine LMS. Other reports showed that among the most intriguing changes in genes were losses of JAK1 (1p31-p32) and PSMB9/b1i (6p21.3)
Uterine LMS are relatively rare mesenchymal tumours, having an estimated annual incidence of 0.64 per 100 000 women. They account for approximately one-third of uterine sarcomas and 1.3% of all uterine malignancies. They are the disease with extremely poor prognosis, considering aggressive malignancies with a 5-year survival rate of only 50% for tumours confined to the uterus. At present, surgical intervention is virtually the only means of treatment for uterine LMS
IFN-g treatment markedly increased the expression of PSMB9/b1i, a subunit of the proteasome, which alters the proteolytic specificity of proteasomes. Sequence analysis demonstrated that the loss of IFN- g responsiveness in the human uterine LMS cell line was attributable to the inadequate kinase activity of JAK1 due to a G781E somatic mutation in the ATP-binding region
The growth of JAK1-deficient cell lines reportedly is unaffected; similarly, the cell cycle distribution pattern of freshly explanted tumour cells derived from JAK1-deficient tumours shows no response to IFN-g signaling
The down regulation of major histocompatibility complex (MHC) expression, including the
A total of 51 patients aged between 32 and 83 years who were diagnosed with smooth muscle tumours in the uterus were selected from pathological files. Serial sections were cut from at least 2 tissue blocks from each patient for hematoxylin and eosin staining and immunostaining. All tissues were used with the approval of the Ethical Committee of Shinshu University after obtaining written consent from each patient. The pathological diagnosis of human uterine mesenchymal tumours was performed using established criteria with some modifications
Immunohistochemical staining for PSMB9/b1i, EstrogenReceptor (ER), Progesterone Receptor (PR), TP53, and Ki-67/MIB1 was performed on the serial human uterine LMS sections. Antibodies for ER(ER1D5), PR(PR10A), TP53(DO-1), and Ki-67(MIB-1) were purchased from Immunotech (Marseille, France). Anti-human PSMB9 antibody was produced by SIGMA-Aldrich collaboration Laboratory (SIGMA-Aldrich, Japan Science and Technology Agency (JST) and Shinshu University). IHC was performed using the avidin-biotin complex method previously described. Briefly, one representative 5-mm tissue section was cut from a paraffin-embedded sample of the radical hysterectomy specimen from patients with uterine LMS. Sections were deparaffinized and rehydrated in graded alcohols and then incubated with normal mouse serum for 20 min. Sections were incubated at room temperature for 1 h with primary antibody. Afterwards, sections were incubated with a biotinylated secondary antibody (Dako, Carpinteria, CA, USA) and then exposed to a streptavidin complex (Dako). Complete reaction was revealed by 3, 3¢-diaminobenzidine, and the slide was counterstained with hematoxylin. Normal USM portions in the specimens were used as positive controls. Negative controls consisted of tissue sections also incubated with normal rabbit IgG instead of the primary antibody. These studies are registered, at Shinshu University in accordance with local guidelines (approval no. M192)
The expressions
Equal amounts of proteins (20 mg) were size fractionated on 7.5% SDS–polyacrylamide gel electrophoresis and transferred onto a poly-vinylidene difluoride membrane (PVDF). The blots were allowed to air dry and then placed in blocking buffer (1% BSA in 10 mM Tris buffer with 100 mM NaCl, 0.1% Tween-20, pH 7.5) for 1 h at room temperature. The blots were then incubated with specific primary antibodies for 1 h at room temperature. All the primary antibodies were mouse monoclonal or rabbit polyclonal, obtained from several industries, and were used at different final dilutions (1:1000~1:500) in the blocking buffer. These antibodies were raised using the following proteins as immunogens: PSMB9/b1i (23.4 kDa protein), PSMB8/b5i (30.3 kDa protein), b-ACTIN (41.7 kDa protein). The blots were washed three times for 30 min each with wash buffer (10 mM Tris, 100 mM NaCl, 0.1% Tween-20, pH 7.5) and then incubated with alkaliphosphatase conjugated goat-anti-mouse IgG antibody or anti-rabbit IgG antibody (Promega, Madison, WI) diluted in 5% non-fat milk in wash buffer. The PVDF membranes were washed with wash buffer three times for 30 min, and Western Blue Stabilized Substrate (Promega Co. Madison, WI) was added and incubated as previously reported
To demonstrate whether the somatic mutations in the ATP-binding region and kinase activation domain of the JAK1 molecule, promoter region of
We sincerely appreciate the generous donation of PSMB9/b1i-deficient breeding mice and technical comments by Dr. Susumu Tonegawa, Massachusetts Institute of Technology. We thank Isamu Ishiwata for his generous gift of the uterine LMS cell lines. This work was supported by grants from the Ministry of Education, Culture, Science and Technology, the Japan Science and Technology Agency, the Foundation for the Promotion of Cancer Research, Kanzawa Medical Research Foundation, and The Ichiro Kanehara Foundation.
To determine whether somatic mutations exist in the ATP-binding region or kinase activation domain of JAK 1 and JAK2, in the promoter region of PSMB9/b1i gene at Tyr701 or Ser727 of STAT1, or in the ATP-binding region and kinase activation domain of JAK2 in human uterine LMS, genomic DNA was isolated and direct sequencing was carried out. Genomic DNA was extracted from consecutive paraffin-embedded human uterine LMS tissue and normal myometrium tissue sections using the microwave-based DNA extraction method for PCR amplification(1). To avoid contamination of normal myometrium or inflammatory cells, the tumour areas were confirmed using a hematoxylin and eosin-stained glass slide as a template. The tumour tissues were scraped by razor-micro dissection from paraffin-embedded consecutive tissue sections. The genomic DNA was subjected to PCR, and restricted DNA fragments for direct sequencing analysis were amplified using published oligonucleotide primers. PCR products were directly sequenced using a DYEnamic Terminator Cycle Sequencing Kit (Amersham-Biosciences, Piscataway, NJ) with an ABI Prism 3100 Genetic Analyzer (Applied Biosystem, Foster City, CA). The sequences of mutant JAK1, STAT1, and the promoter region of PSMB9/b1i gene derived from individual uterine LMS tissue sections are registered in the DDBJ (Accession: AB219242, DJ055380, DJ055379, DJ055378, DJ055377, DJ055376).
1. Banerjee SK, Makdisi WF, Weston AP, et al. Microwave-based DNA extraction from paraffin-embedded tissue for PCR amplification. BioTechniques 1995; 18: 768-774.
JAK1:(F, 5’-caccaaatctttaaaccggaccccagcctt-3’, R, 5’-tacgatggggcttccctgataacagcacat-3’),(F, 5’-atggcttt ctgtgctaaaatgaggagctcc-3’, R, 5’-tccatcctgctcggtcttggggtctcgaat-3’), (F, 5’-attcgagaccccaagaccgagcagga tgga-3’, R, 5’-tccactggattccaagattcccagtcacca-3’), (F, 5’-tggtgactgggaatcttggaatccagtgga-3’, R, 5’-ggcg gctcatgaggtctcccaagctgggga-3’), (F, 5’-tccccagcttgggagacctcatgagccacc-3’, R, 5’-ccgtaatggggatgccggg gtcactgagct-3’), and (F, 5’-agctcagtgaccccggcatccccattacgg-3’, R, 5’-cagatcagctatgtggttacctccactctc-3’)
JAK2:(F, 5’-cagattatgggtaatgattaaaggctccca-3’, R, 5’-cacagcatttctccaacatctgacaaccaaacc-3’), (F, 5’-ga cagtctgctaattccagctactagaa-3’, R, 5’-gcctctccctctgggcattggcataagtcc-3’), and F, 5’-atgaagcaaccgtgttga agtagacattag-3’, R, 5’-cccacgtggactataaccatgactataagacc-3’), Primer sets for the nested-PCR: (F, 5’-gaa actatttgagtttccctgtatcatttag-3’, R, 5’-ctacaagcactccttaaaatgttgtagaaag-3’),(F, 5’-gtaatttgccttgaaaactggt atttcc-3’, R, 5’-gcataagtccagatcgttaagacattgtac-3’), and (F, 5’-gaagtagacattaggaaatcatctagacg-3’, R, 5’-cactgttactgtaaatatagaaatggcaaac-3’)
STAT1: (Ser727 F, 5’-cacttattgagagctacacacaggccagcc-3’, R, 5’-ggctggggacatgagaatcccatgagctgt-3’) and (Tyr701 F, 5’-tgctgataggcagtaacacggggatctcaa-3’, R, 5’-aggaggctaagctgtct agaaacacagtag-3’) Primer sets for the nested-PCR:
(Ser727 F, 5’-ttgagagctacacacaggccagccgtggta-3’, R, 5’-gggacatgagaatcccat gagctgtacttt-3’) and (Tyr701 F, 5’-tgctgataggcagtaacacggggatctcaa-3’, R, 5’-gtctagaaacacagtagaacttt aatcccc-3’)
The promoter region of PSMB9/b1i gene: (F, 5’-cgagaagctcagccatttaggggaaagcga-3’, R, 5’-cgcccgcagc atccctgcaaggcaccgctc-3’). Primer sets for the nested-PCR: (F, 5’-aagcgaaatcgaaagcggccgcctgctcac-3’, R, 5’-ctctcctcgccgcctggggcactggtttcc-3’)