Biomarkers and Applications(ISSN: 2576-9588)

research article

  PDF Download

Cessation of Dormancy Involves an Increase of the Expression of TGFβ1 at Protein Level in Breast Cancer

Kristiina Joensuu1*, Marja Heiskala1, Päivi Heikkilä1, Marjut Leidenius2

1Department of Pathology and HUSLAB, Helsinki University Hospital and University of Helsinki, FIN-00290 Helsinki, Finland

2Department of Breast Surgery, Comprehensive Cancer Center Helsinki University Hospital, P.O Box 163, 00290 HUS, Helsinki, Finland University of Helsinki, Central Hospital, FIN-00290 Helsinki, Finland

*Corresponding author: Kristiina Joensuu, Department of Pathology and HUSLAB, Helsinki University Hospital and University of Helsinki, FIN-00290 Helsinki, Finland

Received Date: 29 December, 2020; Accepted Date: 06 January, 2021; Published Date: 14 January, 2021

Abstract

The duration of dormancy of breast cancer after removing the primary tumor is unpredictable, and depends on the properties of the malignant epithelial cells and on the immunological response of the host. In this study, we investigated the role of TGFβ signalling in tumor progression by estimating the expression TGFβ1, TGFβ2 and TGFβ3 at protein level in primary tumours and the corresponding recurrences. The frequency of Foxp3 transcription factor positive T cells was also evaluated. We used 137 paraffin fixed samples of primary breast cancer and their corresponding recurrent lesions, first recorded 0-2, 5-10 and >10 years after the diagnosis. The results showed that the expression of TGFβ1was significantly higher in the recurrent lesions than in the primary tumors, regardless of tumor type and state. The frequency of intratumoral Foxp3 positive lymphocytes was associated with the tumor cell expression of TGFβ1 in the primary tumors. The protein level expression of none of the TGFβ isoforms nor Foxp3 predicted the duration of the dormancy. In primary tumors TGFβ1 associated with lobular histology, ER and PR positivity and a low grade, TGFβ2 with a smaller tumor size, and TGFβ3 with ductal type of the tumor, a high expression of Ki67 and HER2 positivity.

Keywords

Breast cancer dormancy; TGFβ-signalling

Introduction

Tumor dormancy after surgical treatment and individually designed adjuvant therapy of breast cancer depends on interactions between the remaining malignant cells and the immune response of the host. We have previously shown that a high expression of AZIN1 in tumor cells as well as in the microenvironment [1], and an activation of the CCL2 pathway in the tumor stroma [2], predict an early recurrence. In order to further investigate the transition of early (primary) breast cancer into a disseminated disease, we examined the protein level expression of TGFβ1, 2 and 3, and the frequency of Foxp3 positive T cells in primary tumors recurring at three different time points (early, intermittent, late), and in their corresponding recurrent or metastatic lesions.

TGFβ belongs to a large family of proteins with activity on cell growth and differentation, including the activin/inhibin subfamily, the Bone Morphogenetic Proteins (BMPs), nodal, myostatin, and the Mullerian Inhibitory Substance (MIS) [3- 6]. Three distinct isoforms of TGFβ have been identified in mammalian cells (TGFβ-1, -2, -3), each encoded by a different gene [7-10]. Cell function regulation by TGFβ is conducted via an interaction with cell surface receptors I, II, III (TβRI, II, III) [11- 14]. Both TGFβ and its receptors are widely expressed in all cell types, and defects in TGFβ signaling are associated with various human diseases from autoimmunity to cancer. The active TGFβ molecule, synthesized from a large inactive precursor molecule, the latent TGFβ, is a homodimer stabilized by hydrophobic interactions strengthened by a disulfide bond [15].

The role of TGFβ signaling in controlling the life of normal and malignant cells is dual: in normal cells and early cancer it inhibits cell proliferation and immortalization and induces apoptosis, whereas in aggressive cancers the signaling via TGFβRII becomes defective because of genetic and epigenetic modifications in the TGFβ signaling components, or because cells become resistant to the signaling, due to the activation of pro-oncogenic signaling pathways (MAPK, PI3 K, Ras, c-MYC), which override the growth inhibitory signaling pathways, including TGFβ/Smad [16-19]. The tumor suppressive functions are then replaced by induction of Epithelial-Mesenchymal Transition (EMT), loss of cell adhesion, increased migration, invasion, chemo attraction, and tumor metastasis [20-25]. An important part of the pro-tumoral effects of the TGFβ pathway consists of the capability to mediate and modify tumor-stroma interactions and to remodel tumor microenvironment [26]. The role of TGFβ signaling in the induction of Foxp3+ regulatory T cells has recently been intensively investigated, and simultaneously a direct, Foxp3- independent TGFβ-mediated function regulating auto reactive T cells and maintaining peripheral T cell tolerance has also been recognised [27].

The impact of TGFβ signalling has primarily been evaluated based on observations on the TGFβ1 isoform, whereas few articles describe the potentially different roles of the other TGFβ isoforms. The isoforms seem to have different functions during mammary gland development and involution [28-31], and different patterns of expression have also been reported in different types of cancer. A higher expression of TGFβ1 and TGFβ3 has been reported in breast cancer versus normal tissue, with no difference in the expression of TGFβ2 [32,33]. TGF isoforms have also been reported to have distinct patterns of co-expression with TGFβ receptors, depending on the type of cancer.

In this study we evaluated the role of TGFβ signaling in tumor dormancy and progression in breast cancer by investigating the expression of TGFβ1, TGFβ2 and TGFβ3 at protein level in the primary tumors with different prognoses. We also compared their expression in primary tumors versus corresponding recurrent or metastatic lesions. To follow the possible immunomodulating impact of the TGFβ pathway, we investigated the frequency of Foxp3+ T cells in the same set of samples.

Materials and Methods

Patients and tissue samples

Paraffin-embedded tissue blocks from Primary Breast Tumors (PTs) of 137 patients and their corresponding Recurrent Lesions (Rs) were collected from the archives of the Department of Pathology, Helsinki University Hospital as described previously [34]. The primary tumors had been removed in 1974-2006. The cases were selected based on the time lapse from the primary operation to the first recurrence to represent quickly (≥2 years), intermediately (5-10 years) and slowly (>10 years) progressing tumors. All consecutive cases fulfilling the requirement of the treatment-relapse interval were recruited, and three groups were formed: Group 1 n=41, tumors with relapse (R) within two years after primary surgery, Group 2 n= 57, with R after 5 -10 years and Group 3 n=39 with R after > 10 years (range 10 to 23 years). The archival slides were re-examined, and the histological tumour type and grade were assigned based on the criteria of Elston and Ellis [35]. The clinico pathologic characteristics of the patients and their cancers are summarized in Table 1. The Ethical Committee of the Helsinki University Central Hospital approved the study protocol.

Immunohistochemistry

The protein level expression of the three human isoforms of the cytokine TGFβ, TGFβ1, TGFβ2, TGFβ3, and the transcription factor Fork Head Box P3 (Foxp3) was investigated in the samples, and the grading of the expression level was based on viewing the entire tumor sections at the 20x magnification. The type of the marker-positive cells was specified using the 40x magnification. For TGFβ1, TGFβ2 and TGFβ3 the expression level in tumor cells was scored as 0: no antigen expression, +1: 5-29% of antigen expressing cells, +2: 30-69% of antigen expressing cells, and +3: 70- 100% of antigen expressing cells. The evaluation of the frequency of Foxp3 expressing lymphocytes was performed according to Sobottka et al. [36], with tuFoxp3 representing intratumoral Foxp3 positive lymphocytes in direct contact with tumor cells, and strFoxp3 representing Foxp3 positive lymphocytes within stromal areas of the invasive tumor. The frequency was scored as negative: no Foxp3 expressing lymphocytes, +1: 5-29% of lymphocytes expressing Foxp3, +2: 30-69% of lymphocytes expressing Foxp3, and +3: 70-100% of lymphocytes expressing Foxp3.

The staining results for ER, PR, HER2 and MIB1 was evaluated according to a standard protocol [37,38]. All tumors with an over expression of HER2 at protein level [34,38] were tested for HER2 gene amplification by Inform HER2 Dual ISH test (Inform HER2 Dual In Situ Hybridization) [39].

Four μm thick sections were deparaffinised in xylene and rehydrated. To block endogenous peroxidase, the slides were treated in a PT module (LabVision UK Ltd.Suffolk, UK) in TrisHCL buffer (ph 8,5) for 20 min at 98°C and with 0,3% Dako REAL Peroxidase Blocking Solution for 15 min. Immunostaining was performed in an Auto stainer 480 (Lab Vision Thermo scientific, UK Ltd.Cheshire, UK) by addition of the primary antibodies, see below, followed by 30 min incubation with Dako RealEnVision/ HRP detection system, Rabbit/Mouse (ENV) reagent (Dako, K5007), and the visualization of staining was done by REAL DAB+Chromogen (Dako, K5007) for 10 min. Washing with PBS0, 04%-Tween20 took place between each step. Both staining’s were counterstained with Mayer`s hematoxylin and mounted in mounting medium.

As primary antibodies the following reagents were used: polyclonal rabbit anti TGFβ1, anti TGFβ2, and anti TGFβ3 antibodies at dilution 1:200, 1:500 and 1:500, respectively, (Spring Bioscience USA), mouse monoclonal anti Foxp3 clone 236A/E7 at dilution 1:500, (Abcam United Kingdom), mouse monoclonal anti-ER alfa clone 6F11, at dilution 1:50 (Novo Castra Newcastle United Kingdom), anti-PR alfa clone 636, at dilution 1:100 (DacoCytomation Denmark), anti-HER2 clone CB11, at dilution 1:400 (Novo Castra United Kingdom), and anti-Ki67 clone MIB1, dilution 1:75 (Daco Cytomation Denmark).

Normal human placenta tissue was used as positive control for TGFβ1, TGFβ2 and TGFβ3, and normal human tonsil tissue for Foxp3.

The samples were analysed independently by three different pathologists (KJ, MH, PH) for scoring, and a partial re-evaluation confirmed the reproducibility of the results.

Statistical methods

All statistical analyses were performed using SPSS 24.0 for Windows (SPSS Incorporation, Chicago, IL, USA). The differences between the expression of the markers in PTs and the corresponding Rs within the Groups were tested using the paired samples t-test. Kruskal-Wallis test and the Mann-Whitney U test was used for comparing differences between the Groups. For analyzing the association of the expression of the markers with the Clinicopathologic parameters, ER, PR, Ki67 and HER2 we used the categorical two-tailed Pearson’s chi-square test. For Pearson’s Chi-square tests, the cut-off point for negativity versus positivity for TGFβ1, TGFβ2 and TGFβ3 was 0 versus ≥1, and for Foxp3 < 2 versus ≥ 2. For ER and PR the cut-off point for positivity was 1%, and for Ki67 ≥14%. Regarding HER2, only the tumors with a positive gene amplification were considered HER2 positive. Probability values p<0.05 were considered significant except in the Mann-Whitney U test, where P < 0,0167 (< 0,5/3) was used.

Results

TGFβ1

In the entire tumor set TGFβ1 was widely expressed; the frequency of TGFβ1 positive tumor cells was ≥1 in 59 (43,1% ) of the Pts and in 111 (81,0%) of the Rs. The TGFβ1 positivity was high (≥2+) in 10 (7,3%) cases of the Pts and in 26 (19%) of the Rs. The protein was evenly distributed in the cytoplasm and nucleus (Figure 1).

The mean expression TGFβ1 at protein level was significantly higher in the Rs as compared to the corresponding Pts in the whole set of samples (p=0.0001). The difference was significant also in all the three Groups analysed separately; the p-value was 0.001 in Group 1, 0.0001 in Group 2 and 0.001 in Group 3 (Figure 2). When compared to each other, the Groups showed the same level of TGFβ1 expression; time to relapse was not reflected in the expression in the Pts nor in the corresponding Rs (Figure 2).

The expression of TGFβ1 (> 0) correlated positively with the expression of ER (p=0.074), and PR (p=0.055), although the result did not reach statistical significance. The other conventional prognostic markers of breast cancer, Ki67 and HER2, did not show correlation. The expression of TGFβ1 was more often negative in ductal than in lobular breast cancer (p=0.046), and in high grade (2-3) than in low-grade (1) tumors (p=0.014). TGFβ1 positivity versus negativity did not correlate with the nodal status (Table 2).

TGFβ2

TGFβ2 was widely expressed in the entire tumor set; the frequency of TGFβ2 positive tumor cells was ≥1 in 119 (86,9%) of the Pts and 117 (85,4%) of the Rs. The expression in tumor cells was high (≥2+) in 85 (62,0%) cases of PTs and 92 (67,2%) in Rs. Notably, the expression of TGFβ2 in the epithelial tumor cells was higher than that of TGF-β1 and TGFβ3, and the subcellular distribution of the protein was typical for TGFβ2, the samples showing granular cytoplasmic and frequently prominent cell membrane-associated staining (Figure 1 and 2).

The expression of TGFβ2 did not significantly vary between the PTs and the Rs when looking at the whole set of samples. When analysing the Groups separately, a higher expression in Rs than in the corresponding Pts was shown in Group 3 (p=0.010).

There was a significant difference in TGFβ2 mean expression between the Rs of the three Groups (p=0.0001, Kruskal-Wallis test, data not shown). Analysed by the Mann-Whitney test the difference was significant between the Groups 2 and 3, the expression being higher in the Group 3 (p=0,0001 (<0,167, <0,5/3), data not shown).

A high expression of TGFβ2 associated with the lobular histological type of the tumor (p=0,018), and TGFβ2 negative tumors were larger (>20 mm) than those showing TGFβ2 expression (p=0.052), but no correlation was shown to the other conventional prognostic markers of breast cancer, nor to the stage of the tumor (Table 2).

TGFβ3

In the entire tumor set TGFβ3 was widely expressed; the frequency of TGFβ3 positive cells was ≥1 in 109 (79,6%) of the Pts and in 117 (85,4%) in the Rs. The expression was high (≥2) in 40 (29,2%) PTs and 43 (31,4%) in Rs. The protein was evenly distributed in the cytoplasm and nucleus (Figure 1).

The expression levels of TGFβ3 did not differ significantly between the PTs and the Rs in the entire tumor set nor in the Groups analysed separately. There were no significant differences in the expression of TGFβ3 between the Groups, neither with respect to the PTs nor the Rs.

A high expression of TGFβ3 tended to associate with HER2 over expression (p=0.056), and TGFβ3 negativity associated with a low expression of Ki67 (p=0.029), but no correlation was shown to other conventional prognostic markers of breast cancer, nor to the stage of the tumor (Table 2).

Foxp3

In the entire set of samples Foxp3 positive lymphocytes infiltrated the tumors in 87 (63, 5%) of the PTs and in 94 (68, 6%) of the Rs, and the stroma in 97 (70, 8%) of the PTs and in 93 (67, 9%) of the Rs. A high frequency (≥2) of Foxp3 positive lymphocytes was seen intratumorally in 36 (26, 3%) of the Pts and in 28 (20, 4%) of the Rs, and stromally in 16 (11, 7%) of the Pts and in 18 (13, 1%) of the Rs. The frequency of Foxp3 positive lymphocytes was similar in Pts and in their corresponding Rs both in the entire set of samples, and in the Groups analysed separately (Figure 3).

A high expression of intratumoral Foxp3 tended to be associated with ER and PR positivity (p-values 0.007 and 0.068, respectively) and with the lobular histology of the tumor (p=0.002). A low expression of intratumoral Foxp3 associated with TGFβ1 negativity, and with a high (2-3) tumor grade (p=0,029) (Table 2).

A high expression of stromal Foxp3 (strFoxp3) associated with a high Ki67 positivity (p=0,005), a larger tumor size (< 20mm, p=0,020) and with the ductal type of the tumor (p=0,022) (Table 2).

Discussion

The impact of the TGFβ signaling in cancer has primarily been investigated based on observations on TGFβ1, whereas the role of the other TGFβ isoforms remains uncertain. The three TGFβ isomers have been shown to have different functions during mammary gland development and involution [28-31], suggesting that their impact on tumor development is different as well. In this study our aim was to determine the correlation of the protein level expression of the three TGFβ isoforms and Foxp3 with the duration of dormancy after the treatment of primary breast cancer, and to compare the expression levels in primary tumors versus the corresponding recurrent and metastatic lesions to evaluate their role in tumor progression.

TGFβ1

Our results indicated that immunoreactive TGFβ1 was found in the majority of the samples from primary invasive breast cancer, and the expression level was significantly higher in the corresponding metastatic lesions. We found no significant correlation between the mean levels of TGFβ1 protein expression and ER, PR, HER2, histology, aggressiveness nor nodal status, whereas when comparing TGFβ1 negative tumors (0) versus those expressing TGFβ1 (≥1) we saw TGFβ1 to be associated with some of the favorable prognostic markers (ER and PR expression, low grade of tumor). This is in agreement with the current consensus of TGFβ protein expression correlating in early-stage tumors with a favorable prognosis, and in advanced tumors with tumor aggressiveness and poor prognosis [32,40-44]. We saw a uniform cytoplasmic staining in cancer cells, whereas previously, secreted TGFβ1 has been shown to strongly localize to the advancing edge of the tumor [44]. The difference in choosing epitopes for immunization to produce antibodies against TGFβ1 may explain the diversity of the results. In our study TGFβ1 negative tumors were more of the ductal than lobular histology, the latter known to have a less favorable prognosis [45]. This finding might reflect a different mechanism of tumor induction in ductal versus lobular breast cancer. Moreover, while tumor type-dependent differences in the mRNA level expression of TGFβ1 have previously been reported, a clear non-accordance between TGFβ1 mRNA and protein levels has been noted, too, and this makes the interpretation of the findings complicated and method-dependent [33,46]. In our material the level of the TGFβ1 protein expression in primary tumors did not differ between the Groups with different times to progression, meaning that it had no prognostic value. Understandably the complexity of activating the latent TGFβ1 protein, and the dual role of TGFβ1 in the progression of cancer make the mere presence of immunoreactive TGFβ1 an insufficient marker to monitor the biological behavior of the tumor.

TGFβ2

In our study TGFβ2 was widely expressed both in primary breast cancer and in recurrences. The signal for the protein was over all stronger than that of TGFβ1 and TGFβ3. The expression level was similar in Pts in all the Groups, indicating that time to progression had no impact. Pts and Rs did not differ from each other except for the Group 3, where the expression was significantly higher in Rs than in Pts. In Rs, the expression tended to be higher in the Groups with a long dormancy. In Group 3 it was significantly higher than in Group 2. We also noted an association of a high expression of TGFβ2 with lobular histology of breast cancer.

Higher levels of TGFβ1 and TGFβ3 mRNA expression have been shown in cancer patients compared to normal tissues, and in more advanced tumors compared to early stage tumors, with no significant changes in TGFβ2 expression [32,33]. High TGFβ2 mRNA levels in breast tumours have been reported to predict a better better prognosis especially in lymph node-negative diseases [32]. However, an up-regulated TGFβ2 mRNA level in advanced compared to early tumors has also been reported, and an inverse correlation between TGFβ2 protein and mRNA has been documented [47]. Our observation of a tendency to an increase of TGFβ2 at protein level in metastatic lesions in late recurring breast cancer is new, and suggests an active role for TGFβ2 in the process of the termination of tumor dormancy. Lobular histology is associated with a prognosis inferior to ductal [45], and our observation of a high TGFβ2 expression at protein level in tumors with this histology may have a causal connection. Malignant transformation and progression may differ between the histological subtypes of breast cancer, and may utilize different parts of the TGFβ pathway.

TGFβ3

In our study the expression levels of TGFβ3 did not differ significantly between the Pts and the Rs in the entire tumor set nor in the Groups analysed separately, and the time to progression had no impact on the expression in Pts nor in Rs.

TGFβ3 has an isoform-specific function in embryonic palate fusion and wound healing [48-50], but its possible role in tumorigenesis and tumor progression has only sporadically been studied. A high expression of TGFβ3 at mRNA level has been noted in breast tumors with a high stage, the protein level, however, remaining elusive (32). Elevated levels of TGFβ3 expression have been reported in late-stage tumours [51], and in glioblastomas a high expression of TGFβ3 has been reported to correlate with a poor prognosis [52]. An experimental study in these tumors has suggested that TGFβ3 might function as a gatekeeper controlling downstream signaling despite high expression of TGFβ1 and TGFβ2 isoforms [53]. Our result showed a connection between a high protein level expression of TGFβ3 and two adverse prognostic markers, HER2 and a high expression of Ki67, but no independent predictive value regarding the time to progression was detected. The result is in agreement with the so far acquired information of the complexity of the TGFβ signalling, the mere amount of immune reactive TGFβ isoform not necessarily illustrating the level of the activity of the pathway.

Foxp3

In our study, Foxp3 was widely expressed in lymphocytes in direct contact with tumor cells (intratumoral) and infiltrating the surrounding stroma (stromal) with no difference in prevalence between primary tumors and recurrent or metastatic lesions, nor between the Groups with different times to progression. Hence, our result showing no predictive value as for the time to progression is in accordance with the previous data showing variable association with good or poor prognosis depending on the tumor type [54]. The mechanisms maintaining peripheral T cell tolerance overlap with those involved in tumor-induced development of its micro environment, and the transcription factor Foxp3 is crucial for the process. It can be induced in the thymus at the presence of interleukin-2 (IL-2), or in the peripheral immune system under the influence of TGFβ and IL-2 [55,56]. In our study the number of intratumoral Foxp3 positive lymphocytes followed the expression of TGFβ1 in the cancer cells, suggesting that in early breast cancer the TGFβ pathway, clearly involved in the development of the tumor micro environment [57], may also be the primary inducer of Foxp3 positive Tregs.

Our results show a significantly increased expression of TGFβ1 in recurrent lesions of breast cancer as compared to the corresponding primary tumor, suggesting that TGFβ pathway is an important factor in the transition of local breast cancer into a disseminated disease, where the option of complete cure is lost. An adjuvant treatment blocking the activation of TGFβ pathway should be considered at the time of the removal of the primary lesion.

Acknowledgements

We thank Eija Heiliö for her excellent technical assistance, and Antti Nevanlinna, Msc, for fascilitating the statistical analysis.

Funding: Helsinki University Central Hospital Research Foundation.

Availability of Data: The datasets generated during and/ or analysed during the current study are available from the corresponding author on reasonable request.

Ethics Approval: The Ethics Committee of Helsinki University Central Hospital approved the study protocol.

Authors’ contributions

Kristiina Joensuu: Planning the study, responsible for the collection and handling of the samples, evaluating immunohistochemical stainings, making the statistical work, writing the article.

Marja Heiskala: Doing the literature search, planning the study, evaluating the samples, writing the article, preparing the graphics.

Marjut Leidenius: Critical review of the article.

Päivi Heikkilä: Planning the study, analysing the stainings.


Figure 1: Expression of TGFβ1, TGFβ2 and TGFβ3 in a sample of Pt of breast cancer and in its corresponding R lesion after 9 years; immunohistochemical staining at 40x magnification.


Figure 2: Mean expression levels of TGFβ1, TGFβ2 and TGFβ3 in primary tumors and corresponding recurrent lesions in the entire set of 137 pairs of samples, and separately analysed in Groups 1, 2 and 3 with short, intermediate and long time, respectively, to progression. The expression levels were determined by IHC.


Figure 3: Mean frequencies of Foxp3 positive intratumoral and stromal lymphocytes in primary tumors and corresponding recurrent lesions in the entire set of 137 pairs of samples, and separately analysed in Groups 1, 2 and 3 with short, intermediate and long time, respectively, to progression.

 

Group 1 n=41

Group 2 n=57

Group 3 n=39

Age at surgery of  primary tumor (PT)

< 50 years

19

20

18

50 years

22

37

21

Tumor size

20 mm

14

28

24

< 20 mm

26

28

15

system missing

1

1

 

Lymph node

negative

14

34

21

positive

system missing

24

3

20

3

13

5

Grade

1

4

7

8

2

22

35

26

3

15

15

5

 

 

 

 

Histological type

ductal

24

36

16

lobular

17

19

23

special types

0

2

0

 

Tissue site of recurrence (R)

 

skin

6

10

11

soft tissue

6

12

5

subcutaneous tissue

12

16

15

lung

0

4

2

brain

2

2

0

lymph node

2

1

2

ovary

0

1

0

bone

3

6

4

liver

5

2

0

pleura

0

1

0

peritoneum

2

1

0

mesenterium

1

0

1

larynx

1

0

0

uterus

1

0

1

duodenum

0

1

0

NOTE. In  Group 1 recurrences were detected within two years, in Group 2 from 5 to 10 years and in Group 3  >10 years after primary surgery. R was defined as any local or regional recurrence or any distant metastatic disease. Adapted from ref. 2 where the same set of tumor samples was used.


Table 1: Clinicopathologic parameters of the 137 breast cancer patients and the site of recurrence.

 

PR negative

PR positive

p

TGFβ 1 negative

25 (37,9%)

41 (62,1%)

 

TGFβ 1positive

13 (22,0%)

46 (78,0%)

0,055

 

ductal type

lobular type

 

TGFβ 1 negative

42 (62,7%)

25 (37,3%)

 

TGFβ 1positive

26 (44,8%)

32 (55,2%)

0,046

 

low grade

high grade

 

TGFβ 1 negative

4 (5,9%)

64 (94,1%)

 

TGFβ 1 positive

12 (20,3%)

47 (79,7%)

0,014

 

FoxP3 low

FoxP3 high

 

TGFβ 1 negative

56 (86,2%)

9 (13,8%

 

TGFβ 1 positive

30 (52,6%)

27 (47,4%)

0,0001

 

size < 20 mm

size 20 mm

 

TGFβ 2 negative

3 (100%)

0 (0%)

 

TGFβ 2 positive

51 (43,6%)

66 (56,4%)

0,052

 

Ki67 negative, <14%

Ki67 positive, 14%

 

TGFβ 3 negative

11 (84,6%)

2 (15,4%)

 

TGFβ 3 positive

56 (52,8%)

50 (47,2%)

0,029

 

ducatal type

lobular type

 

iFoxP3 low

54 (62,8%)

32 (37,2%)

 

iFoxP3 high

11 (31,4%)

24 (68,6%)

0,002

 

low grade

high grade

 

iFoxP3 low

7 (8,0%)

80 (92,0%)

 

iFoxP3 high

8 (22,2%)

28 (77,8%)

0,029

 

ER negative

ER positive

 

iFoxP3 low

37 (43,0%)

49 (57,0%)

 

iFoxP3 high

6 (17,1%)

29 (82,9%)

0, 007

 

size < 20 mm

size20 mm

 

sFoxP3 low

48 (50,0%)

48 (50,0%)

 

sFoxP3 high

3 (18,8%)

13 (81,3%)

0,020

 

ductal type

lobular type

 

sFoxP3 low

48 (50,5%)

47 (49,5%)

 

sFoxP3 high

13 (81,3%)

3 (18,8%)

0,022

 

Ki67 negative, <14%

Ki67 positive,14%

 

sFoxP3 low

59 (62,8%)

35 (37,2%)

 

sFoxP3 high

4 (25,0%)

12 (75,0%)

0,005


Table 2: Relationship between TGFβ1, TGFβ2, TGFβ3 and Foxp3 with ER, PR, Ki67 IHC expression and HER2 gene amplification in the 137 primary tumors of breast cancer. The categorical Pearson's chi-squared test (χ2) was used. Only significant results are shown.

References

  1. Joensuu K, Heiskala M, Leidenius M, Heikkilä P (2019) A High Expression of AZIN1 but not AZIN2 is involved in the progression of Breast Cancer. J Cancer Biol Clin Oncol 2: 1-8.
  2. Heiskala M, Leidenius M, Joensuu K, Heikkilä P (2019) High expression of CCL2 in tumor cells and abundant infiltration with CD14 positive macrophages predict early relapse in breast cancer. Virchows Archiv 474: 3-12.
  3. Massagué J (2008)TGFβ in ca Cell 134: 215-230.
  4. Lebrun JJ (2009) Activin, TGF-β and menin in pituitary tumorigenesis. Advances in Experimental Medicine and Biology 668: 69-78.
  5. Humbert L, Neel JC, Lebrun JJ (2010) Targeting TGF-beta signaling in human cancer therapy. Trends in Cell & Molecular Biology 5: 69-107.
  6. Lebrun JJ, Chen Y, Vale WW (1997) Receptor serine kinases and signaling by activins and inhibins. In Aono T, Sugino H Vale W W (eds) Inhibin, Activin and Follistatin. Serono Symposia USA, Springer, New York, NY.
  7. Massagué J (2008) TGFβ in cancer. Cell 134: 215-230.
  8. Massagué J (1998) TGF-β signal transduction. Ann Rev Biochem 67: 53-791.
  9. Derynck R, Akhurst J, Balmain A (2001) TGF-β signaling in tumor suppression and cancer pr Nature Genetics 29:117-129.
  10. Chin D, Boyle GM, Parsons PG, Coman WB (2004) What is transforming growth factor-beta (TGF-β)? Brit J Plastic Surgery 57: 215-221.
  11. Massague J, Cheifetz S, Laiho M, Ralph DA, Weis FM, et al. (1992) Transforming growth factor-beta. Cancer Surv 12: 81-103.
  12. Massague J, Attisano L, Wrana JL (1994) The TGF-beta family and its composite receptors. Trends Cell Biol 4: 172-178.
  13. Massague J (1998) TGF-beta signal transduction. Annu Rev Biochem  67: 753-791.
  14. Hata A, Shi Y, Massague J (1998) TGF-beta signaling and cancer: structural and functional consequences of mutations in Smads. Mol Med Today 4: 257-262.
  15. Sun PD, Davies DR (1995) The cystine-knot growth-factor superfamily. Annu Rev Biophysics and Biomolecular Structure 24: 269-291.
  16. Derynck R, Akhurst RJ, Balmain A (2001) TGF-β signaling in tumor suppression and cancer pro Nature Genetics 29: 117-129.
  17. Wakefield LM, Roberts AB (2002) TGF-β signaling: positive and negative effects on tumo Current Opinion in Genetics and Development 12: 22-29.
  18. Nagata H, Hatano E, Tada M, Murata M, Kitamura K, et al. (2009) Inhibition of c-Jun NH 2 terminal kinase switches Smad3 signaling from oncogenesis to tumor-suppression in rat hepatocellular carcinoma. Hepatology 49: 1944-1953.
  19. Akhurst RJ, Derynck R (2001) TGF-β signaling in cancer-a double-edged swor Trends in Cell Biology 11: S44-S51.
  20. Humbert L, Neel JC, Lebrun JJ (2010) Targeting TGF-beta signaling in human cancer ther Trends in Cell & Molecular Biology 5: 69-107.
  21. Xu J, Lamouille S, Derynck R (2009) TGF-β-induced epithelial to mesenchymal transition Cell Res 19:156-172.
  22. Their JP (2002) Epithelial-mesenchymal transitions in tumor progression. Nature Reviews Cancer 2: 442-454.
  23. Yingling JM, Blanchard KL, Sawyer JS (2004) Development of TGF-β signalling inhibitors for cancer therapy. Nature Reviews Drug Discovery 3:1011-1021.
  24. Siegel PM, Massagué J (2003) Cytostatic and apoptotic actions of TGF-β in homeostasis and can Nature Reviews Cancer 3: 807-821.
  25. Dumont N, Arteaga CL (2003) Targeting the TGFβ signaling network in human neoplasia. Cancer Cell 3: 531-536.
  26. Neuzillet C, de Gramont A, Tijeras-Raballand A, de Mestier L, Cros J, et al. (2014) Perspectives of TGF-beta inhibition in pancreatic and hepatocellular carcinomas. Oncotarget 5: 78-94.
  27. Oh SA, Liu M, Nixon BG, Kang D, Toure A, et al. (2017) Foxp3-independent mechanism by which TGF-β controls peripheral T cell tolerance. PNAS E7540.
  28. Robinson SD, Silberstein GB, Roberts AB, Flanders KC, Daniel CW (1991) Regulated expression and growth inhibitory effects of transforming growth factor-beta isoforms in mouse mammary gland development. Development 113: 867-878.
  29. Strange R, Li F, Saurer S, Burkhardt A, Friis RR (1992) Apoptotic cell death and tissue remodelling during mouse mammary gland involution. Development 115: 49-58.
  30. Nguyen AV, Pollard JW (2000) Transforming growth factor beta3 induces cell death during the first stage of mammary gland involution. Development 127: 3107-3118.
  31. Monks J (2007) TGF beta as a potential mediator of progesterone action in the mammary gland of pregnancy. Mammary Gland Biol Neoplasia 12: 249-257.
  32. Hachim MY, Hachim IY, Dai M, Ali S, Lebrun JJ (2018) Differential expression of TGFb isoforms in breast cancer highlights different roles during breast cancer progression. Tumor Biology 1-12.
  33. Chen C, Zhao KN, Masci PP, Lakhani SR, Antonsson A, et al. (2015) TGFβ isoforms and receptors mRNA expression in breast tumours: prognostic value and clinical implications. BMC Cancer 15: 1010-1022.
  34. Joensuu K, Heikkilä P, Andersson LC  (2008) Tumor dormancy: elevated expression of stanniocalcins in late relapsing breast cancer. Cancer Lett 265: 76-83.
  35. Elston CW , Ellis IO (1991) Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology 19: 403-4
  36. Sobottka B, Pestalozzzi B, Fink D, Moch H, Varga Z (2016) Similar lymphocytic infiltration pattern in primary breast cancer and their corresponding distant metastases. Oncoimmunology 5: e1153208.
  37. Hammond MEH, Hayes DF, Dowsett M, Allred DC, Hagerty KL, et al. (2010) American Society of Clinical Oncology/College of American pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast ca Arch Pathol Lab Med 134: 907-922.
  38. Wolff AC, Hammond ME, Schwartz JN (2007) American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol 25: 118-145.
  39. Joensuu K, Leidenius M, Kero M, Andersson LC, Horwitz KB, et al. (2013) ER, PR, HER2, Ki-67 and CK5 in Early and Late Relapsing Breast Cancer-Reduced CK5 Expression in Metastases. Breast Cancer (Auckl) 7: 23-34.
  40. Reiss M (1997) Transforming growth factor-beta and cancer: a love-hate relationship? Oncol Res 9: 447-457.
  41. Gold LI (1999) The role for transforming growth factor (TGF-b) in human cancer. Crit Rev Oncog 10: 303-360.
  42. Wang Y, Liu T, Tang W, Deng B, Chen Y, et al. (2016) Hepatocellular carcinoma cells induce regulatory T cells and lead to poor prognosis via production of transforming growth factor β Cell Physiol Biochem 38: 306-318.
  43. Gorsch SM, Memoli VA, Stukel TA, Gold LI, Arrick BA (1992) Immunohistochemical staining for transforming growth factor β1 associates with disease progression in human breast cancer. Cancer Res 52: 6949-6952.
  44. Dalal BI, Keown PA, Greenberg AH (1993) Immunocytochemical localization of secreted transforming growth factor-β1 to the advancing edges of primary tumors and to lymph node metastases of human mammary carcinoma. Am J Pathol 143: 381-389.
  45. Chen Z, Yang J, Li S, Lv M, Shen Y, et (2017) Invasive lobular carcinoma of the breast: A special histological type compared with invasive ductal carcinoma. PLOS ONE.
  46. Soufla G, Porichis F, Sourvinos G, Vassilaros S, Spandidos DA (2006) Transcriptional deregulation of VEGF, FGF2, TGF-b1, 2, 3 and cognate receptors in breast tumorigenesis. Cancer Lett 235: 100-113.
  47. Dave H, Trivedi S, Shah M, Shukla S (2011) Transforming growth factor beta 2: a predictive marker for breast cancer. Indian J Exp Biol 49: 879-887.
  48. Taya Y, O'Kane S, Ferguson MW (1999) Pathogenesis of cleft palate in TGF-beta3 knockout mice. Development 126: 3869-3879.
  49. Yang LT, Kaartinen V (2007)TGFbTgfb1 expressed in the Tgfb3 locus partially rescues the cleft palate phenotype of Tgfb3 null mutants. Dev Biol 312: 384-395.
  50. Shah M, Foreman DM, Ferguson MW (1995) Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring. J Cell Sci 108: 985-1002.
  51. Laverty HG, Wakefield LM, Occleston NL, O’Kane S, Ferguson MW (2009) TGF-beta3 and cancer: a review. Cytokine Growth Factor Rev 20: 301-317.
  52. Frei K, Gramatzki D, Schroeder JJ, Espinoza I, Rushing EJ, et al. (2015) Transforming growth factor-beta pathway activity in gliob Onco-target 6: 5963-5977.
  53. Seystahl K, Papachristodouluo A, Burghardt I, Schneider H, Hasenbach K, et al. (2017) Biological Role of Therapeutic Targeting of TGF-β3 in Glioblas Moleculr Cancer Ther 16: 1177-1185.
  54. Martin F, Ladoire S, Mignot G, Apetoh L, Ghiringhelli F (2010) Human FOXP3 and cancer. Oncogene 29: 4121-4129.
  55. Mueller DL (2010) Mechanisms maintaining peripheral tolerance. Nat Immunol 11: 21-27.
  56. Hori S, Nomura T, Sakaguchi S (2003) Control of regulatory T cell development by the transcription factor Foxp3. Science 299: 1057-1061.
  57. Papageorgis P, Stylianopoulos T (2015) Role of TGFβ in regulation of the tumor microenvironment and drug delivery (Review). Int J Oncol 46: 933-943.

Copyright and Licensing: This is an Open Access Journal Article Published Under Attribution-Share Alike CC BY-SA: Creative Commons Attribution-Share Alike 4.0 International License. With this license readers can share, distribute, download, even commercially, as long as the original source is properly cited. Read More.

   

share article