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Article

Breast Cancer and p16: Role in Proliferation, Malignant Transformation and Progression

by
Dalibor V. Jovanovic
1,
Slobodanka L. Mitrovic
1,*,
Milos Z. Milosavljevic
2,
Milena B. Ilic
1,
Vesna D. Stankovic
1,
Milena S. Vuletic
1,
Milica N. Dimitrijevic Stojanovic
1,
Danijela B. Milosev
2,
Goran L. Azanjac
3,
Vladica M. Nedeljkovic
4 and
Dragce Radovanovic
5
1
Department of Pathology, Faculty of Medical Sciences, University of Kragujevac, 34000 Kragujevac, Serbia
2
Department of Pathology, University Medical Centre Kragujevac, 34000 Kragujevac, Serbia
3
Department of Plastic Surgery, University Medical Centre Kragujevac, 34000 Kragujevac, Serbia
4
Institute of Pathology, Faculty of Medicine, University in Pristina—Kosovska Mitrovica,38220 Kosovska Mitrovica, Serbia
5
Department of Surgery, Faculty of Medical Sciences, University of Kragujevac, 34000 Kragujevac, Serbia
*
Author to whom correspondence should be addressed.
Healthcare 2021, 9(9), 1240; https://doi.org/10.3390/healthcare9091240
Submission received: 26 May 2021 / Revised: 4 September 2021 / Accepted: 6 September 2021 / Published: 21 September 2021
Browse Figures
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Abstract

:
The definition of new molecular biomarkers could provide a more reliable approach in predicting the prognosis of invasive breast cancers (IBC). The aim of this study is to analyze the expression of p16 protein in IBC, as well as its participation in malignant transformation. The study included 147 patients diagnosed with IBC. The presence of non-invasive lesions (NIL) was noted in each IBC and surrounding tissue. p16 expression was determined by reading the percentage of nuclear and/or cytoplasmic expression in epithelial cells of IBC and NIL, but also in stromal fibroblasts. Results showed that expression of p16 increases with the progression of cytological changes in the epithelium; it is significantly higher in IBC compared to NIL (p < 0.0005). Cytoplasmic p16 expression is more prevalent in IBC (76.6%), as opposed to nuclear staining, which is characteristic of most NIL (21.1%). There is a difference in p16 expression between different molecular subtypes of IBC (p = 0.025). In the group of p16 positive tumors, pronounced mononuclear infiltrates (p = 0.047) and increased expression of p16 in stromal fibroblasts (p = 0.044) were noted. In conclusion, p16 protein plays an important role in proliferation, malignant transformation, as well as in progression from NIL to IBC.

1. Introduction

Invasive breast cancer (IBC) is a heterogeneous group of tumors that show different biological behavior, prognosis, and response to treatment [1]. Numerous studies have investigated the association of individual biomarkers with clinicopathological characteristics and/or treatment outcomes [2,3,4]. Despite the significance of prognostic factors, their accuracy in assessing disease outcomes and determining therapeutic strategies is limited. Therefore, the definition and validation of new molecular biomarkers could provide a more reliable approach in predicting the prognosis of this disease, which would indicate the possibility of application for therapeutic purposes.
Literature data indicate the importance of cell cycle dysregulation in malignant transformation. Cell cycle regulation is controlled by a number of factors, of which p16 protein is one of the most important. p16 (p16INK4a or cyclin-dependent kinase 2A inhibitor) is a protein that blocks cell cycle progression from G1 to S phase by inhibiting the activity of cyclin-dependent kinase complexes 4 and 6 (CDK4/6)/cyclin D1 (CCND1) [5,6]. CCND1/CDK4/6 normally phosphorylates retinoblastoma protein (pRb), its inhibition results in a hypophosphorylated form of pRb, which binds E2F transcription factors and leads to cell cycle arrest and transcription inhibition [7,8,9].
Constant activation of p16, which is most often the result of DNA damage, leads to permanent cell cycle arrest and senescence [10,11,12]. Cell senescence is an irreversible cell cycle arrest in the G0 phase, resulting from telomere shortening or due to oncogenic stress, without the possibility DNA synthesis after mitogenic stimuli [13]. The irreversibility of senescence suggests the assumption that it could be one of the factors that suppress malignant transformation and tumor progression [14]. One of the senescence forms can be started with oncogene activation, which results in the so-called oncogene-induced senescence (OIS). It results in the activation of a number of antioncogenes, which are responsible for the introduction and maintenance of the cell in the G0 phase. One of the most important molecules of OIS is p16, which is considered a tumor suppressor due to its physiological role and reports on its role in the origin and development of IBC are contradictory [15,16,17,18,19,20,21].
The aim of this study is to analyze the expression of p16 protein in IBC, as well as its participation in malignant transformation.

2. Materials and Methods

2.1. Study Design

The study, approved by the decision of the Ethics Committee number 01/17/2290, included 147 patients with a diagnosis of breast cancer, who were diagnosed and treated at the University Clinical Center in Kragujevac, Serbia in the period from 2012 to 2017.
Pathohistological analysis of tumors on H&E stained specimens was performed on the operative material obtained by tumerectomy, quadrantectomy and/or mastectomy with dissection of regional lymph nodes [22]. The presence of non-invasive lesions (NIL) [in situ lobular and ductal carcinoma (ISC), lobular and ductal atypical hyperplasia (AH) and normal ductal and acinar epithelium (NE)] was noted in each IBC and surrounding tissue. Based on microscopic analysis, IBCs were classified into three groups: ductal, lobular, and a group of other histological types. E-cadherin expression differentiated ductal from lobular breast cancer. At the same time, all relevant macroscopic, pathohistological and prognostic parameters (tumor size, histological type and grade, nodal status, presence of necrosis, intra and peritumoral mononuclear infiltrate, perineural, lymphatic and vascular invasion, molecular subtype IBC and disease stage) were defined [23].
IBCs are classified according to the recommendations into four molecular groups: Luminal A, Luminal B, HER2+, and TNBC [24].

2.2. Immunohistochemical (IHC) Procedure

Tissue sections were fixed in 10% neutral buffered and stabilized formalin solution, pH 7.0 and embedded into paraffin. IHC was performed on a tissue section of a representative paraffin block of each patient. Tissue sections 4 μm thick were applied to adherent slides (SuperFrost® Plus, VWR, Leuven, Belgium), then deparaffinized in xylene and rehydrated in decreasing alcohol concentrations. After epitope retrieval, endogenous peroxidase was blocked with 3% hydrogen peroxide for 5 min. The preparations were incubated with primary monoclonal and polyclonal antibodies at room temperature for the recommended duration. The following antibodies were used, ready for use or in appropriate dilution: p16INK4a antibody (CINtec Histology Kit, ROCHE, Mannheim, Germany), mAb ER (1D5, RTU, IR657, DAKO, Gloustrup, Denmark), mAb PR (PgR636, RTU, IR068, DAKO, Gloustrup, Denmark), pAb HER2 (1:1200, AO485, DAKO, Gloustrup, Denmark), Ki67 (1:200, MIB-1, IR626, DAKO, Gloustrup, Denmark). After washing the primary antibody, tissue sections were incubated with commercial biotinized secondary antibody at room temperature, for the recommended duration (En Vision FLEX HRP, RTU, K8000). The IHC reaction was visualized using 3,3′-diaminobenzidine tetrahydrochloride (DAB). The preparations were finally contrasted with Mayer’s hematoxylin (Hematoxylin M, HEMM-OT-1L, Biognost, Croatia), and the coverslips were mounted with Canada balsam. Tissue samples in which incubation with the primary antibody was omitted were used as negative controls of the IHC reaction, and IBCs with known expression of the analyzed markers were used as positive controls. Slides were analyzed at 100×, 200×, and 400× magnifications, using a light microscope (AxioScop 40, Carl Zeiss, Germany). Representative sections were photographed using a digital camera (AxioCam ICc1, Carl Zeiss, Germany).

2.3. Evaluation of IHC Staining

Immunohistochemical stains were rated by two independent pathologists, who were blinded to clinical follow-up data at the time of analysis. In cases of differently assessed expression, agreement was reached by joint analysis of the preparation and consultation with a third pathologist.
Analysis of estrogen (ER) and progesterone receptors (PR) expression was performed using Allred score [25] as the sum of the percentage of positive nuclei of tumor cells and the intensity of IHC staining. The Allred score ranges from 0 to 8.
The expression of human epidermal growth factor receptor 2 (HER2) was analyzed based on standard recommendations [26]. Depending on the continuity and intensity of membrane staining, all IBCs were classified into HER2 negative (0 and 1+) and HER2 positive (3+). Equivocal HER2 (2+) was retested by silver in situ hybridization (SISH) technique after which patients were classified as HER2 positive or negative IBCs.
Ki67 IHC expression was defined as the percentage of positive tumor cells per 100 counted in the zone of highest tumor proliferation. According to the previously defined limit value of Ki67 expression in our laboratory, IBCs were classified into 3 groups: low (Ki67 < 15%), medium (Ki67: 15–30%), and high proliferative activity (Ki67 > 30%) [24,27,28].
p16 expression was determined as the percentage of nuclear and/or cytoplasmic expression in IBC and NIL epithelial cells, but also in stromal fibroblasts. By analyzing the expression, we defined the cutoff value for p16. Based on the obtained result, we divided all IBCs into p16 positive and p16 negative. At the same time, p16 expression was analyzed in stromal fibroblasts and classified into: negative (<10%), low (10–24%), medium (25–50%), high (>50% positive cells) [29]. Presence of intra and peritumor mononuclear infiltrate was defined as: absent (0%), low (<30%), medium (30–60%), and high (>60%) [30].

2.4. Statistical Data Processing

The commercial software package SPSS (version 22.0, SRSS Inc., Chicago, IL, USA) was used for statistical processing of the data obtained. In the analysis of the obtained results we used: descriptive statistics methods, Man–Whitney test, Kruskal–Wallis test, χ2 test, Pearson or Spearman correlation coefficient, ROC curve (with determination of cutoff value, sensitivity and specificity). By determining the sensitivity and specificity of the test, the level of practical reliability of statistical analysis was determined. All reported p values were 2-sided and p < 0.05 was considered statistically significant.

3. Results

3.1. General Characteristics

Our study included 147 women with IBC, with an average age of 58 years (range 29–84). Simultaneously with IBC, ISC (ductal or lobular) was present in 79 patients, in 82 AH, and in 109 cases NE were noted. The average cancer size was 22.5 mm (range 9–68 mm). Clinicopathological characteristics are summarized in Table 1.

3.2. p16 Expression Increases with the Progression of Cytological Changes in the Epithelium

The average value of p16 expression increases from NE, through AH and ISC to IBC and is in NE 2.5%, in AH 10%, in ISC 30%, and in IBC 50%. The expression of p16 is significantly higher in IBC compared to NIL (Figure 1A). Furthermore, p16 expression in IBC cells increases with expression of the same marker in NIL cells. Using the Spearman correlation coefficient, we found a strong positive correlation between p16 expressions in all of these changes (Figure 1B–G). The histomorphological picture of various changes with p16 expression is shown in Figure 1H–K.

3.3. The Expression of p16 in Tumor Cells Depend on the Molecular Subtype of IBC

IBCs are classified into molecular subtypes in accordance with the recommendations [24]. Their representation in our sample is shown in Table 2.
There is a difference in p16 expression between different molecular subtypes of IBC (Figure 2A). There is no difference in p16 expression depending on the expression of HER2 and Ki67 (Figure 2B,C). However, with increasing p16 expression in tumor cells, the expression of ER and PR decreases significantly, respectively (Figure 2D,E). The IHC expression of p16 in individual molecular subtypes is shown in Figure 2F–I.

3.4. p16 Expression Is a Marker of Breast Tumor Progression

As presented in Figure 3, our results and the obtained ROC (receiver operating characteristic) curve, indicate that increased p16 expression may be a reliable marker of IBC progression (area under the receiver operating characteristic, AUROC = 0.770; p < 0.0005).
Also, the results show that a cutoff value of 17.5% of p16 positive tumor cells allows obviously separation of NIL patients from IBC patients (sensitivity 0.754, specificity 0.638). Statistical analysis confirmed that increased p16 expression indicates malignant transformation of lesions in the breast.
To analyze the association of p16 expression with parameters indicating tumor progression, we applied the cutoff values of our calculation and IBCs were divided into the group with negative (≤17.5%) and positive p16 expression (>17.5%). Further, our study showed that in the group of p16 positive IBCs, increased expression of p16 in stromal fibroblasts was observed (Figure 4A) and increased presence of mononuclear infiltrate (Figure 4B).
Furthermore, we found an association between p16 positive IBCs and examined clinicopathological characteristics (Table 3).

3.5. Tumor Invasiveness Depends on the Subcellular Localization of p16 Protein Expression

Cytoplasmic expression of p16 may indicate tumor progression, as opposed to nuclear staining, which is characteristic of most noninvasive changes (Table 4).
Dominant nuclear expression of p16 present in NE and AH, in contrast to cytoplasmic expression in ISC and IBC where it was highest (Figure 5).
The percentage of tumors with cytoplasmic expression of p16 was significantly higher in the group of tumors with necrosis present and pronounced mononuclear infiltration (Table 5). In addition, a higher degree of p16 positive fibroblasts was found in the same group of tumors, although the difference was not statistically significant (Table 5). Furthermore, the subcellular localization of p16 was not associated with other clinicopathological parameters (results not shown).

4. Discussion

Molecular profiling has provided evidence of breast tumor heterogeneity, and there is therefore a continuing interest in identifying new markers that will help predict prognosis and response to therapy.
In our study, we examined the expression of p16 protein in the tissue of 147 patients with IBC in whose environment the fields of ISC, AH, and normal ductoacinar structures of the breast parenchyma are present. The results of the study showed that the expression increases from NE, through AH and ISC to IBC. Consistent with the fact that p16 tumor is a suppressor protein, its expression increases with increasing epithelial cell proliferation, as a natural response of tumor suppressor to resist uncontrolled growth. In such circumstances, expression should reach its maximum in ISC changes, followed by a plateau in IBC, indicating a role for p16 in preventing malignant transformation of preinvasive changes in the breast [31].
Benign tumors overexpress p16, which inhibits cell proliferation in response to oncogenic stimuli, protecting cells from malignant transformation. However, by activating alternative signaling pathways, p16-dependent tumor suppression, in some cases, will not exhibit adequate function resulting in uncontrolled cell proliferation [32,33]. Based on this, p16 expression may have diagnostic significance in the differentiation of premalignant and malignant lesions [34]. OIS has a strong antitumor role, which has been shown in various benign lesions such as nevus, neurofibroma, and schwannoma, and is accompanied by overexpression of p16 and cell cycle arrest [35,36,37,38,39,40]. In contrast, malignant forms of these tumors show weak or completely absent p16 immunoreactivity which is associated with malignant transformation and progression [38,39,40,41,42].
The results of our study show significantly higher p16 expression in the IBC group than in NIL. A progressive increase in p16 expression from normal to neoplastic tissue has been described in a number of different malignancies [43,44,45,46,47,48,49]. A similar pattern has been observed in skin where p16 expression increases from low levels in premalignant lesions (actinic keratosis) to high levels of expression in in situ and invasive cancers [50]. In addition, increased nuclear expression of p16 has been shown in premalignant and malignant gallbladder lesions, compared with normal epithelium [51]. By studying cytological changes in the squamous epithelium of the cervix, a significantly higher expression of p16 in invasive carcinoma was demonstrated compared to expression in low and high squamous intraepithelial dysplastics lesions, and it was suggested that this marker could be used to differentiate noninvasive from invasive cervical changes [52,53].
Rahmawati Pare et al. show higher expression of p16 in invasive cancer compared to NIL in the breast [31]. The results of this and other studies are consistent with ours, and show that increased p16 expression in malignant breast tissue correlates with negative ER and PR status [31,44,54,55]. In addition, we indicate that increased p16 expression is accompanied by an increased proliferation index determined by Ki67 nuclear expression, as reported by other authors [56,57]. These results indirectly show that high p16 expression assumes a poorer prognosis of the disease [56,57]. The p16 protein is present in the G1 phase of the cell cycle and has a very long half-life, which is why it accumulates in cells with an increase in the number of divisions. This may partly explain the higher p16 expression in highly proliferative tumor cells than in normal breast epithelial cells, and may also be the reason for the positive correlation between p16 expression and Ki67. Gauthier et al. have shown that women with a pathohistological diagnosis of ISC who express high p16 and high Ki67 develop a new tumor, and accordingly Ki67 classifies high p16 expression into two groups: those who develop subsequent breast cancer and another group of women who do not. Tumors that develop after ISC with high p16/high Ki67 expression are often invasive breast cancer. The remaining ISCs showing high p16 immunopositivity had low Ki67, indicating that these cells retained regulation of the p16/Rb checkpoint. Indeed, most lesions exhibiting a high p16/low Ki67 expression phenotype were not associated with ISC recurrence or IBC occurrence [58]. In the Kerlikowske trial, p16 was the only individual marker correlating with an invasive recurrence after ISC [59].
Our analysis of molecular types of IBC showed that HER2+ and TNBC have the highest expression of p16. Shin et al. reported an association of high p16 expression with Luminal B and HER2+ tumors [60] while other studies presented results similar to ours, particularly regarding the association of high p16 expression in TNBC [61,62,63,64].
In different benign and malignant changes, it has been observed that p16 expression may occur in the nucleus and/or cytoplasm simultaneously, suggesting that different localization of p16 has a different role in the process of proliferation and tumorigenesis [65]. By analyzing the results of our study, we observed an increasing trend of epithelial cytoplasmic expression of p16 from NE, via AH and ISC to IBC, where expression was also the highest, in contrast to nuclear expression which was highest in NE and progressively decreased towards IBC. Numerous studies have attempted to explain the importance of different subcellular localization of the p16 protein. The regulation of the cell cycle, as the most important function of p16, takes place in the nucleus. On the other hand, cytoplasmic staining in some tumors indicated tumor progression and poor prognosis [66]. For example, in breast tumors, p16 expression was limited to the nucleus in fibroadenoma, and nuclear-cytoplasmic or exclusively cytoplasmic expression was observed in cancer [44,45]. Cytoplasmic expression of p16 can be explained by the formation of the p16 complex with sequestered CDK4, which cannot pass through the nuclear membrane and consequently accumulates in the cytoplasm [46,67,68]. Furthermore, its localization may depend on post-translational modifications or its ability to form a complex with other proteins [69]. p16 interacts with various proteins in the cytoplasm, e.g., with α-β-γ actin and α-β tubulin which play a role in cell migration and cell cytoskeleton construction [70]. Another protein associated with aberrant accumulation of p16 in the cytoplasm is anion exchanger 1 (AE1) [71]. The interaction of transmembrane protein AE1 and p16 in gastric and colon carcinomas results in sequestration of p16 and leads to the accumulation of both proteins in the cytoplasm [71]. In addition, Shen et al. show that decreased AE1 function induces the release and transition of p16 from the cytoplasm to the nucleus, leading to cell death and inhibition of tumor growth [71]. Because of all of the above, localization of p16 in the cytoplasm may represent an alternative mechanism for modulating different signaling pathways, rather than a simple way to inactivate the cell cycle control function [67].
A significant result of our study is that in p16-positive IBCs, there is a more pronounced intra and peritumor mononuclear infiltrate, as well as higher p16 expression in stromal fibroblasts. A group of authors examined p16 expression in the stroma of cervical mucinous carcinoma and concluded that overexpression of p16 in stromal cells contributes to the growth, progression, and more aggressive behavior of this tumor [72]. Published results suggest that the p16 protein promotes invasiveness through interactions with other molecules associated with tumor cell migration [67,73,74,75,76]. Overexpression of p16 in tumor cells and stromal fibroblasts contributes to tumor progression by secreting proinflammatory cytokines (IL-1, IL-6 and IL-8) and proteases (e.g., matrix metalloproteinases) [77,78,79]. Prolonged presence of such cells maintains a chronic inflammatory microenvironment that is essentially tumorigenic [80,81,82,83]. Further, it modulates other important features of cancer by local facilitation of neovascularization [84], epithelial-mesenchymal transition [80,85], tumor invasion [86,87], and cell plasticity [88]. p16-positive stromal fibroblasts produce molecules that can promote tumor development in vivo and malignant phenotype formation in cell culture models. These effects have been observed in a large number of cell types, including breast tumor cell lines [80,89,90,91,92], skin [93], prostate [80,94], pancreas [95], and oro-pharyngeal mucosa [96]. One of the most immediate, protumorogenic effects is to promote epithelial cell proliferation. In the case of breast parenchyma, stromal fibroblasts stimulate the proliferation of premalignant and malignant epithelial cells [80,89,91]. It has also been shown that human lung stromal fibroblasts can promote tumor growth, stimulating angiogenesis by secreting increased concentrations of proangiogenic factors, one of which is vascular endothelial growth factor [84]. All of the above indicates that p16 expression in stromal fibroblasts and more pronounced mononuclear infiltrate affect the growth and progression of various neoplasms, including IBC.
Regarding standard biomarkers in all cases of IBC, hormonal status (ER and PR), Ki-67 proliferative index and HER2 are determined, in order to define the prognosis and establish therapeutic possibilities, including hormone therapy, chemotherapy, and anti-HER2 therapy. However, unlike p16, these markers cannot tell us the difference between NIL and IBC. In the Kerlikowske trial, p16 was the only individual marker correlating with an invasive recurrence after ISC [59]. Special importance should be given to TNBC tumors due to the lack of targeted therapy [97]. Earlier studies performed on the animal model IBC and on material of human origin confirmed an increase in p16 expression in the TNBC group, in particular, basal-like tumor subtypes. Standard markers for determining the basal type phenotype are EGFR, CK5/6 and CK14, and these studies have shown their association with p16 expression [63,98,99].
In this way, the importance of p16 expression in the group of basal-like breast cancer was confirmed. However, the disadvantage of these studies is the reading of p16 expression in the nucleus and cytoplasm, which we tried to compensate by a special analysis of the subcellular localization of p16. Our study opens the door to future research, where special attention would be paid to the association of subcellular localization of p16 and TNBC.

5. Conclusions

Our results confirmed that increased p16 expression indicates malignant transformation of changes in the breast and progression of IBC. A cutoff value of p16-positive tumor cells allows the separation of patients with NIL from those with IBC, so that IHC analysis of p16 expression can be used as an additional diagnostic test in separating benign from malignant changes in the breast. Also, excessive cytoplasmic and loss of nuclear expression of p16 are very important in the evolution of tumors from NE to IBC and indicate tumor progression. All of the above could recommend this molecular marker for use for diagnostic purposes or as a therapeutic target.

Author Contributions

D.V.J.: wrote the manuscript; S.L.M., V.D.S., M.Z.M.: performed the pathohistological and statistical analysis and drafting of the manuscript; M.B.I., M.S.V., M.N.D.S., V.M.N.: made PubMed research and assisted in the writing of the manuscript; D.R., D.B.M., G.L.A.: evaluated critically the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of University Clinical Centre Kragujevac (protocol code 01/17/2290; 14 June 2017.)

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Turashvili, G.; Brogi, E. Tumor Heterogeneity in Breast Cancer. Front. Med. 2017, 4, 227. [Google Scholar] [CrossRef] [Green Version]
  2. Ragab, H.M.; Samy, N.; Afify, M.; El Maksoud, N.A.; Shaaban, H.M. Assessment of Ki-67 as a potential biomarker in patients with breast cancer. J. Genet. Eng. Biotechnol. 2018, 16, 479–484. [Google Scholar] [CrossRef] [PubMed]
  3. Hsu, Y.L.; Yen, M.C.; Chang, W.A.; Tsai, P.H.; Pan, Y.C.; Liao, S.H.; Kuo, P.L. CXCL17-derived CD11b(+) Gr-1(+) myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB. Breast Cancer Res. 2019, 21, 23. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Cho, T.M.; Kim, J.Y.; Kim, Y.J.; Sung, D.; Oh, E.; Jang, S.; Farrand, L.; Hoang, V.H.; Nguyen, C.T.; Ann, J.; et al. C-terminal HSP90 inhibitor L80 elicits anti-metastatic effects in triple-negative breast cancer via STAT3 inhibition. Cancer Lett. 2019, 447, 141–153. [Google Scholar] [CrossRef] [PubMed]
  5. Pandey, K.; An, H.J.; Kim, S.K.; Lee, S.A.; Kim, S.; Lim, S.M.; Kim, G.M.; Sohn, J.; Moon, Y.W. Molecular mechanisms of resistance to CDK4/6 inhibitors in breast cancer: A review. Int. J. Cancer 2019, 145, 1179–1188. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Ding, L.; Cao, J.; Lin, W.; Chen, H.; Xiong, X.; Ao, H.; Yu, M.; Lin, J.; Cui, Q. The Roles of Cyclin-Dependent Kinases in Cell-Cycle Progression and Therapeutic Strategies in Human Breast Cancer. Int. J. Mol. Sci. 2020, 21, 1960. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  7. Topacio, B.R.; Zatulovskiy, E.; Cristea, S.; Xie, S.; Tambo, C.S.; Rubin, S.M.; Sage, J.; Kõivomägi, M.; Skotheim, J.M. Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein’s C-Terminal Helix. Mol. Cell. 2019, 74, 758–770.e4. [Google Scholar] [CrossRef] [PubMed]
  8. Nath, S.; Chowdhury, A.; Dey, S.; Roychoudhury, A.; Ganguly, A.; Bhattacharyya, D.; Roychoudhury, S. Deregulation of Rb-E2F1 axis causes chromosomal instability by engaging the transactivation function of Cdc20-anaphase-promoting complex/cyclosome. Mol. Cell Biol. 2015, 35, 356–369. [Google Scholar] [CrossRef] [Green Version]
  9. McNair, C.; Xu, K.; Mandigo, A.C.; Benelli, M.; Leiby, B.; Rodrigue, D.; Lindberg, J.; Gronberg, H.; Crespo, M.; De Laere, B.; et al. Differential impact of RB status on E2F1 reprogramming in human cancer. J. Clin. Investig. 2018, 128, 341–358. [Google Scholar] [CrossRef] [Green Version]
  10. Kumari, R.; Jat, P. Mechanisms of Cellular Senescence: Cell Cycle Arrest and Senescence Associated Secretory Phenotype. Front. Cell Dev. Biol. 2021, 9, 645593. [Google Scholar] [CrossRef]
  11. Calcinotto, A.; Kohli, J.; Zagato, E.; Pellegrin, L.; Demaria, M.; Alimonti, A. Cellular Senescence: Aging, Cancer, and Injury. Physiol. Rev. 2019, 99, 1047–1078. [Google Scholar] [CrossRef] [PubMed]
  12. Pack, L.R.; Daigh, L.H.; Meyer, T. Putting the brakes on the cell cycle: Mechanisms of cellular growth arrest. Curr. Opin. Cell Biol. 2019, 60, 106–113. [Google Scholar] [CrossRef] [PubMed]
  13. Wang, Z.; Gao, J.; Liu, H.; Ohno, Y.; Xu, C. Targeting senescent cells and tumor therapy (Review). Int. J. Mol. Med. 2020, 46, 1603–1610. [Google Scholar] [PubMed]
  14. Zeng, S.; Shen, W.H.; Liu, L. Senescence and Cancer. Cancer Transl. Med. 2018, 4, 70–74. [Google Scholar]
  15. Scott, A.; Bai, F.; Chan, H.L.; Liu, S.; Ma, J.; Slingerland, J.M.; Robbins, D.J.; Capobianco, A.J.; Pei, X.H. p16INK4a suppresses BRCA1-deficient mammary tumorigenesis. Oncotarget 2016, 7, 84496–84507. [Google Scholar] [CrossRef] [Green Version]
  16. Zhao, L.H.; Liu, H.G. Immunohistochemical detection and clinicopathological significance of JARID1B/KDM5B and P16 expression in invasive ductal carcinoma of the breast. Genet. Mol. Res. 2015, 14, 5417–5426. [Google Scholar] [CrossRef]
  17. Gavressea, T.; Kalogeras, K.T.; Koliou, G.A.; Zagouri, F.; Lazaridis, G.; Gogas, H.; Tsigaridas, K.; Kourtas, A.; Petraki, K.; Markopoilos, C.; et al. The Prognostic Value of the Immunohistochemical Expression of Phosphorylated RB and p16 Proteins in Association with Cyclin D1 and the p53 Pathway in a Large Cohort of Patients with Breast Cancer Treated with Taxane-based Adjuvant Chemotherapy. Anticancer Res. 2017, 37, 2947–2957. [Google Scholar]
  18. Peurala, E.; Koivunen, P.; Haapasaari, K.M.; Bloigu, R.; Jukkola-Vuorinen, A. The prognostic significance and value of cyclin D1, CDK4 and p16 in human breast cancer. Breast Cancer Res. 2013, 15, R5. [Google Scholar] [CrossRef] [Green Version]
  19. Harbhajanka, A.; Lamzabi, I.; Bitterman, P.; Reddy, V.B.; Ghai, R.; Gattuso, P. Correlation of p16 Expression on Cancer and Stromal Cells With Clinicopathologic and Immunohistochemical Features of Lobular Breast Carcinoma. Appl. Immunohistochem. Mol. Morphol. 2019, 27, 658–662. [Google Scholar] [CrossRef]
  20. Pare, R.; Soon, P.S.; Shah, A.; Lee, C.S. Differential expression of senescence tumour markers and its implications on survival outcomes of breast cancer patients. PLoS ONE 2019, 14, e0214604. [Google Scholar] [CrossRef]
  21. Wang, L.; Zhan, X.; Shen, X.; Li, M.; Yang, J.; Yu, W.; Chen, H.; Jin, B.; Mao, Z. P16 promotes the growth and mobility potential of breast cancer both in vitro and in vivo: The key role of the activation of IL-6/JAK2/STAT3 signaling. Mol. Cell Biochem. 2018, 446, 137–148. [Google Scholar] [CrossRef] [PubMed]
  22. Suvarna, K.S.; Layton, C.; Bancroft, J.D. (Eds.) Bancroft’s Theory and Practice of Histological Techniques, 8th ed.; Churchill Livingstone/Elsevier Science: London, UK, 2018. [Google Scholar]
  23. Tan, P.H.; Ellis, I.; Allison, K.; Brogi, E.; Fox, S.B.; Lakhani, S.; Lazar, A.J.; Morris, E.A.; Sahin, A.; Salgado, R.; et al. WHO Classification of Tumours Editorial Board. The 2019 World Health Organization classification of tumours of the breast. Histopathology 2020, 77, 181–185. [Google Scholar] [CrossRef]
  24. Goldhirsch, A.; Wood, W.C.; Coates, A.S.; Gelber, R.D.; Thürlimann, B.; Senn, H.J.; Panel members. Strategies for subtypes-dealing with the diversity of breast cancer: Highlights of the St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011. Ann. Oncol. 2011, 22, 1736–1747. [Google Scholar] [CrossRef] [PubMed]
  25. Allred, D.C.; Harvey, J.M.; Berardo, M.; Clark, G.M. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod. Pathol. 1998, 11, 155–168. [Google Scholar] [PubMed]
  26. Wolff, A.C.; Hammond, M.E.; Hicks, D.G.; Dowsett, M.; McShane, L.M.; Allison, K.H.; Allred, D.C.; Bartlett, J.M.; Bilous, M.; Fitzgibbons, P.; et al. American Society of Clinical Oncology; College of American Pathologists Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer. Arch. Pathol. Lab. Med. 2014, 138, 241–256. [Google Scholar] [CrossRef] [Green Version]
  27. Focke, C.M.; Bürger, H.; van Diest, P.J.; Finsterbusch, K.; Gläser, D.; Korsching, E.; Decker, T. German Breast Screening Pathology Initiative. Interlaboratory variability of Ki67 staining in breast cancer. Eur. J. Cancer 2017, 84, 219–227. [Google Scholar] [CrossRef]
  28. Gnant, M.; Thomssen, C.; Harbeck, N. St. Gallen/Vienna 2015: A Brief Summary of the Consensus Discussion. Breast Care 2015, 10, 124–130. [Google Scholar] [CrossRef] [Green Version]
  29. Yoon, N.; Yoon, G.; Park, C.K.; Kim, H.S. Stromal p16 expression is significantly increased in malignant ovarian neoplasms. Oncotarget 2016, 7, 64665–64673. [Google Scholar] [CrossRef] [Green Version]
  30. Schalper, K.A.; Velcheti, V.; Carvajal, D.; Wimberly, H.; Brown, J.; Pusztai, L.; Rimm, D.L. In situ tumor PD-L1 mRNA expression is associated with increased TILs and better outcome in breast carcinomas. Clin. Cancer Res. 2014, 20, 2773–2782. [Google Scholar] [CrossRef] [Green Version]
  31. Pare, R.; Shin, J.S.; Lee, C.S. Increased expression of senescence markers p14(ARF) and p16(INK4a) in breast cancer is associated with an increased risk of disease recurrence and poor survival outcome. Histopathology 2016, 69, 479–491. [Google Scholar] [CrossRef]
  32. Braig, M.; Lee, S.; Loddenkemper, C.; Rudolph, C.; Peters, A.H.; Schlegelberger, B.; Stein, H.; Dörken, B.; Jenuwein, T.; Schmitt, C.A. Oncogene induced senescence as an initial barrier in lymphoma development. Nature 2005, 436, 660–665. [Google Scholar] [CrossRef]
  33. Collado, M.; Blasco, M.A.; Serrano, M. Cellular senescence in cancer and aging. Cell 2007, 130, 223–233. [Google Scholar] [CrossRef] [Green Version]
  34. Inoue, K.; Fry, E.A. Aberrant expression of p16INK4a in human cancers—A new biomarker? Cancer Rep. Rev. 2018, 2. [Google Scholar] [CrossRef] [Green Version]
  35. Michaloglou, C.; Vredeveld, L.C.; Soengas, M.S.; Denoyelle, C.; Kuilman, T.; van der Horst, C.M.; Majoor, D.M.; Shay, J.W.; Mooi, W.J.; Peeper, D.S. BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 2005, 436, 720–724. [Google Scholar] [CrossRef] [PubMed]
  36. Courtois-Cox, S.; Jones, S.L.; Cichowski, K. Many roads lead to oncogene-induced senescence. Oncogene. 2008, 27, 2801–2809. [Google Scholar] [CrossRef] [PubMed]
  37. Krishnamurthy, J.; Torrice, C.; Ramsey, M.R.; Kovalev, G.I.; Al-Regaiey, K.; Su, L.; Sharpless, N.E. Ink4a/Arf expression is a biomarker of aging. J. Clin. Investig. 2004, 114, 1299–1307. [Google Scholar] [CrossRef] [PubMed]
  38. Sargen, M.R.; Merrill, S.L.; Chu, E.Y.; Nathanson, K.L. CDKN2A mutations with p14 loss predisposing to multiple nerve sheath tumours, melanoma, dysplastic naevi and internal malignancies: A case series and review of the literature. Br. J. Dermatol. 2016, 175, 785–789. [Google Scholar] [CrossRef]
  39. Joselow, A.; Lynn, D.; Terzian, T.; Box, N.F. Senescence-Like Phenotypes in HumanNevi. Methods Mol. Biol. 2017, 1534, 175–184. [Google Scholar]
  40. Romagosa, C.; Simonetti, S.; Serrano, C. Senescence markers in Schwannomas. Virchows Arch. 2009, 455, 372. [Google Scholar]
  41. Perrone, F.; Tabano, S.; Colombo, F.; Dagrada, G.; Birindelli, S.; Gronchi, A.; Colecchia, M.; Pierotti, M.A.; Pilotti, S. p15INK4b, p14ARF, and p16 inactivation in sporadic and neurofibromatosis type 1-related malignant peripheral nerve sheath tumors. Clin. Cancer Res. 2003, 9, 4132–4138. [Google Scholar]
  42. Damsky, W.; Micevic, G.; Meeth, K.; Muthusamy, V.; Curley, D.P.; Santhanakrishnan, M.; Erdelyi, I.; Platt, J.T.; Huang, L.; Theodosakis, N.; et al. mTORC1 activation blocks BrafV600E-induced growth arrest but is insufficient for melanoma formation. Cancer Cell 2015, 27, 41–56. [Google Scholar] [CrossRef] [Green Version]
  43. Dai, C.Y.; Furth, E.E.; Mick, R.; Koh, J.; Takayama, T.; Niitsu, Y.; Enders, G.H. p16(INK4a) expression begins early in human colon neoplasia and correlates inversely with markers of cell proliferation. Gastroenterology 2000, 119, 929–942. [Google Scholar] [CrossRef]
  44. Milde-Langosch, K.; Bamberger, A.M.; Rieck, G.; Kelp, B.; Löning, T. Overexpression of the p16 cell cycle inhibitor in breast cancer is associated with a more malignant phenotype. Breast Cancer Res. Treat. 2001, 67, 61–70. [Google Scholar] [CrossRef]
  45. Di Vinci, A.; Perdelli, L.; Banelli, B.; Salvi, S.; Casciano, I.; Gelvi, I.; Allemanni, G.; Margallo, E.; Gatteschi, B.; Romani, M. p16(INK4a) promoter methylation and protein expression in breast fibroadenoma and carcinoma. Int. J. Cancer 2005, 114, 414–421. [Google Scholar] [CrossRef]
  46. Zhao, P.; Mao, X.; Talbot, I.C. Aberrant cytological localization of p16 and CDK4 in colorectal epithelia in the normal adenoma carcinoma sequence. World J. Gastroenterol. 2006, 12, 6391–6396. [Google Scholar] [CrossRef]
  47. Hilliard, N.J.; Krahl, D.; Sellheyer, K. p16 expression differentiates between desmoplastic Spitz nevus and desmoplastic melanoma. J. Cutan. Pathol. 2009, 36, 753–759. [Google Scholar] [CrossRef]
  48. Bai, P.; Xiao, X.; Zou, J.; Cui, L.; Bui Nguyen, T.M.; Liu, J.; Xiao, J.; Chang, B.; Wu, J.; Wang, H. Expression of p14(ARF), p15(INK4b), p16(INK4a) and skp2 increases during esophageal squamous cell cancer progression. Exp. Ther. Med. 2012, 3, 1026–1032. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  49. Zhang, Z.; Rosen, D.G.; Yao, J.L.; Huang, J.; Liu, J. Expression of p14ARF, p15INK4b, p16, and DCR2 increases during prostate cancer progression. Mod. Pathol. 2006, 19, 1339–1343. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  50. Brasanac, D.; Stojkovic-Filipovic, J.; Bosic, M.; Tomanovic, N.; Manojlovic-Gacic, E. Expression of G1/S-cyclins and cyclin-dependent kinase inhibitors in actinic keratosis and squamous cell carcinoma. J. Cutan. Pathol. 2016, 43, 200–210. [Google Scholar] [CrossRef] [PubMed]
  51. Lynch, B.C.; Lathrop, S.L.; Ye, D.; Ma, T.Y.; Cerilli, L.A. Expression of the p16(INK4a) gene product in premalignant and malignant epithelial lesions of the gallbladder. Ann. Diagn. Pathol. 2008, 12, 161–164. [Google Scholar] [CrossRef] [PubMed]
  52. Zouheir, Y.; Fechtali, T.; Elgnaoui, N. Human Papillomavirus Genotyping and p16(INK4a) Expression in Cervical Lesions: A Combined Test to Avoid Cervical Cancer Progression. J. Cancer Prev. 2016, 21, 121–125. [Google Scholar] [CrossRef] [Green Version]
  53. Mendaza, S.; Fernández-Irigoyen, J.; Santamaría, E.; Zudaire, T.; Guarch, R.; Guerrero-Setas, D.; Vidal, A.; Santos-Salas, J.; Matias-Guiu, X.; Ausín, K.; et al. Absence of Nuclear p16 Is a Diagnostic and Independent Prognostic Biomarker in Squamous Cell Carcinoma of the Cervix. Int. J. Mol. Sci. 2020, 21, 2125. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  54. Lebok, P.; Roming, M.; Kluth, M.; Koop, C.; Özden, C.; Taskin, B.; Hussein, K.; Lebeau, A.; Witzel, I.; Wölber, L.; et al. p16 overexpression and 9p21 deletion are linked to unfavorable tumor phenotype in breast cancer. Oncotarget 2016, 7, 81322–81331. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  55. Cui, S.P.; Wang, H.L.; Peng, W.; Liu, H.J.; Hou, L.; Zhang, B. Aberrant expression and correlative analysis of P16 in breast cancers. Beijing Da Xue Xue Bao. 2012, 44, 755–759. [Google Scholar] [PubMed]
  56. Sugianto, J.; Sarode, V.; Peng, Y. Ki-67 expression is increased in p16-expressing triple-negative breast carcinoma and correlates with p16 only in p53-negative tumors. Hum. Pathol. 2014, 45, 802–809. [Google Scholar] [CrossRef] [PubMed]
  57. Subhawong, A.P.; Subhawong, T.; Nassar, H.; Kouprina, N.; Begum, S.; Vang, R.; Westra, W.H.; Argani, P. Most basal-like breast carcinomas demonstrate the same Rb-/p16+ immunophenotype as the HPV-related poorly differentiated squamous cell carcinomas which they resemble morphologically. Am. J. Surg. Pathol. 2009, 33, 163–175. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  58. Gauthier, M.L.; Berman, H.K.; Miller, C.; Kozakiewicz, K.; Chew, K.; Moore, D.; Rabban, J.; Chen, Y.Y.; Kerlikowske, K.; Tlsty, T.D. Abrogated response to cellular stress identifies DCIS associated with subsequent tumor events and defines basal-like breast tumors. Cancer Cell. 2007, 12, 479–491. [Google Scholar] [CrossRef] [Green Version]
  59. Kerlikowske, K.; Molinaro, A.M.; Gauthier, M.L.; Berman, H.K.; Waldman, F.; Bennington, J.; Sanchez, H.; Jimenez, C.; Stewart, K.; Chew, K.; et al. Biomarker Expression and Risk of Subsequent Tumors After Initial Ductal Carcinoma In Situ Diagnosis. J. Natl. Cancer Inst. 2010, 102, 627–637. [Google Scholar] [CrossRef]
  60. Shin, E.; Jung, W.H.; Koo, J.S. Expression of p16 and pRB in invasive breast cancer. Int. J. Clin. Exp. Pathol. 2015, 8, 8209–8217. [Google Scholar]
  61. Ortiz, A.B.; Garcia, D.; Vicente, Y.; Palka, M.; Bellas, C.; Martin, P. Prognostic significance of cyclin D1 protein expression and gene amplification in invasive breast carcinoma. PLoS ONE 2017, 12, e0188068. [Google Scholar] [CrossRef]
  62. Shan, M.; Zhang, X.; Liu, X.; Qin, Y.; Liu, T.; Liu, Y.; Wang, J.; Zhong, Z.; Zhang, Y.; Geng, J.; et al. P16 and p53 play distinct roles in different subtypes of breast cancer. PLoS ONE 2013, 8, e76408. [Google Scholar]
  63. Abou-Bakr, A.A.; Eldweny, H.I. p16 expression correlates with basal-like triple-negative breast carcinoma. Ecancermedicalscience 2013, 7, 317. [Google Scholar]
  64. Bohn, O.L.; Fuertes-Camilo, M.; Navarro, L.; Saldivar, J.; Sanchez-Sosa, S. p16INK4a expression in basal-like breast carcinoma. Int. J. Clin. Exp. Pathol. 2010, 3, 600–607. [Google Scholar] [PubMed]
  65. Kobierzycki, C.; Grzegrzolka, J.; Glatzel-Plucinska, N.; Piotrowska, A.; Wojnar, A.; Smolarz, B.; Romanowicz, H.; Dziegiel, P. Expression of p16 and SATB1 in Invasive Ductal Breast Cancer—A Preliminary Study. In Vivo 2018, 32, 731–736. [Google Scholar] [CrossRef]
  66. Evangelou, K.; Bramis, J.; Peros, I.; Zacharatos, P.; Dasiou-Plakida, D.; Kalogeropoulos, N.; Asimacopoulos, P.J.; Kittas, C.; Marinos, E.; Gorgoulis, V.G. Electron microscopy evidence that cytoplasmic localization of the p16(INK4A) ‘nuclear’ cyclindependent kinase inhibitor (CKI) in tumor cells is specific and not an artifact. A study in non-small cell lung carcinomas. Biotech. Histochem. 2004, 79, 5–10. [Google Scholar] [CrossRef]
  67. Romagosa, C.; Simonetti, S.; López-Vicente, L.; Mazo, A.; Lleonart, M.E.; Castellvi, J.; Ramon y Cajal, S. p16(Ink4a) overexpression in cancer: A tumor suppressor gene associated with senescence and high-grade tumors. Oncogene 2011, 30, 2087–2097. [Google Scholar] [CrossRef] [Green Version]
  68. Gump, J.; Stokoe, D.; McCormick, F. Phosphorylation of p16 correlates with Cdk4 association. J. Biol. Chem. 2003, 278, 6619–6622. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  69. Nilsson, K.; Landberg, G. Subcellular localization, modification and protein complex formation of the cdk-inhibitor p16 in Rb-functional and Rb-inactivated tumor cells. Int. J. Cancer 2006, 118, 1120–1125. [Google Scholar] [CrossRef]
  70. Souza-Rodrigues, E.; Estanyol, J.M.; Friedrich-Heineken, E.; Olmedo, E.; Vera, J.; Canela, N.; Brun, S.; Agell, N.; Hübscher, U.; Bachs, O.; et al. Proteomic analysis of p16 binding proteins. Proteomics 2007, 7, 4102–4111. [Google Scholar] [CrossRef] [PubMed]
  71. Shen, W.W.; Wu, J.; Cai, L.; Liu, B.Y.; Gao, Y.; Chen, G.Q.; Fu, G.H. Expression of anion exchanger 1 sequestrates p16 in the cytoplasm in gastric and colonic adenocarcinoma. Neoplasia 2007, 9, 812–819. [Google Scholar] [CrossRef] [Green Version]
  72. Chung, T.; Do, S.I.; Na, K.; Kim, G.; Jeong, Y.I.; Kim, Y.W.; Kim, H.S. Stromal p16 Overexpression in Gastric-type Mucinous Carcinoma of the Uterine Cervix. Anticancer Res. 2018, 38, 3551–3558. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  73. Jung, A.; Schrauder, M.; Oswald, U.; Knoll, C.; Sellberg, P.; Palmqvist, R.; Niedobitek, G.; Brabletz, T.; Kirchner, T. The invasion front of human colorectal adenocarcinomas shows co-localization of nuclear beta-catenin, cyclin D1, and p16INK4A and is a region of low proliferation. Am. J. Pathol. 2001, 159, 1613–1617. [Google Scholar] [CrossRef]
  74. Natarajan, E.; Saeb, M.; Crum, C.P.; Woo, S.B.; McKee, P.H.; Rheinwald, J.G. Co-expression of p16(INK4A) and laminin 5 gamma2 by microinvasive and superficial squamous cell carcinomas in vivo and by migrating wound and senescent keratinocytes in culture. Am. J. Pathol. 2003, 163, 477–491. [Google Scholar] [CrossRef]
  75. Svensson, S.; Nilsson, K.; Ringberg, A.; Landberg, G. Invade or proliferate? Two contrasting events in malignant behavior governed by p16(INK4a) and an intact Rb pathway illustrated by a model system of basal cell carcinoma. Cancer Res. 2003, 63, 1737–1742. [Google Scholar] [PubMed]
  76. Palmqvist, R.; Rutegard, J.N.; Bozoky, B.; Landberg, G.; Stenling, R. Human colorectal cancers with an intact p16/ cyclin D1/pRb pathway have up-regulated p16 expression and decreased proliferation in small invasive tumor clusters. Am. J. Pathol. 2000, 157, 1947–1953. [Google Scholar] [CrossRef] [Green Version]
  77. Moritani, S.; Ichihara, S.; Hasegawa, M.; Iwakoshi, A.; Murakami, S.; Sato, T.; Okamoto, T.; Mori, Y.; Kuhara, H.; Silverberg, S.G. Stromal p16 expression differentiates endometrial polyp from endometrial hyperplasia. Virchows Arch. 2012, 461, 141–148. [Google Scholar] [CrossRef]
  78. Davalos, A.R.; Coppe, J.P.; Campisi, J.; Desprez, P.Y. Senescent cells as a source of inflammatory factors for tumor progression. Cancer Metastasis Rev. 2010, 29, 273–283. [Google Scholar] [CrossRef] [Green Version]
  79. Coppe, J.P.; Desprez, P.Y.; Krtolica, A.; Campisi, J. The senescence-associated secretory phenotype: The dark side of tumor suppression. Annu. Rev. Pathol. 2010, 5, 99–118. [Google Scholar] [CrossRef] [Green Version]
  80. Coppe, J.P.; Patil, C.K.; Rodier, F.; Sun, Y.; Muñoz, D.P.; Goldstein, J.; Nelson, P.S.; Desprez, P.Y.; Campisi, J. Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol. 2008, 6, 2853–2868. [Google Scholar] [CrossRef]
  81. Orr, F.W.; Wang, H.H. Tumor cell interactions with the microvasculature: A rate-limiting step in metastasis. Surg. Oncol. Clin. N. Am. 2001, 10, 357–381. [Google Scholar] [CrossRef]
  82. Baker, D.J.; Alimirah, F.; van Deursen, J.M.; Campisi, J.; Hildesheim, J. Oncogenic senescence: A multi-functional perspective. Oncotarget 2017, 8, 27661–27672. [Google Scholar] [CrossRef] [Green Version]
  83. Lecot, P.; Alimirah, F.; Desprez, P.Y.; Campisi, J.; Wiley, C. Context-dependent effects of cellular senescence in cancer development. Br. J. Cancer 2016, 114, 1180–1184. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  84. Coppé, J.P.; Kauser, K.; Campisi, J.; Beauséjour, C.M. Secretion of vascular endothelial growth factor by primary human fibroblasts at senescence. J. Biol. Chem. 2006, 281, 29568–29574. [Google Scholar] [CrossRef] [Green Version]
  85. Kuilman, T.; Michaloglou, C.; Vredeveld, L.C.; Douma, S.; van Doorn, R.; Desmet, C.J.; Aarden, L.A.; Mooi, W.J.; Peeper, D.S. Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network. Cell 2008, 133, 1019–1031. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  86. Kim, Y.H.; Choi, Y.W.; Lee, J.; Soh, E.Y.; Kim, J.H.; Park, T.J. Senescent tumor cells lead the collective invasion in thyroid cancer. Nat. Commun. 2017, 8, 15208. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  87. He, Q.; Au, B.; Kulkarni, M.; Shen, Y.; Lim, K.J.; Maimaiti, J.; Wong, C.K.; Luijten, M.N.H.; Chong, H.C.; Lim, E.H.; et al. Chromosomal instability-induced senescence potentiates cell non-autonomous tumourigenic effects. Oncogenesis 2018, 7, 62. [Google Scholar] [CrossRef] [Green Version]
  88. Ritschka, B.; Storer, M.; Mas, A.; Heinzmann, F.; Ortells, M.C.; Morton, J.P.; Sansom, O.J.; Zender, L.; Keyes, W.M. The senescence-associated secretory phenotype induces cellular plasticity and tissue regeneration. Genes Dev. 2017, 31, 172–183. [Google Scholar] [CrossRef] [Green Version]
  89. Krtolica, A.; Parrinello, S.; Lockett, S.; Desprez, P.Y.; Campisi, J. Senescent fibroblasts promote epithelial cell growth and tumorigenesis: A link between cancer and aging. Proc. Natl. Acad. Sci. USA 2001, 98, 12072–12077. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  90. Liu, D.; Hornsby, P.J. Senescent human fibroblasts increase the early growth of xenograft tumors via matrix metalloproteinase secretion. Cancer Res. 2007, 67, 3117–3126. [Google Scholar] [CrossRef] [Green Version]
  91. Parrinello, S.; Coppe, J.P.; Krtolica, A.; Campisi, J. Stromal–epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation. J. Cell Sci. 2005, 118 Pt 3, 485–496. [Google Scholar] [CrossRef] [Green Version]
  92. Tsai, K.K.; Chuang, E.Y.; Little, J.B.; Yuan, Z.M. Cellular mechanisms for low-dose ionizing radiation-induced perturbation of the breast tissue microenvironment. Cancer Res. 2005, 65, 6734–6744. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  93. Sun, P.; Yoshizuka, N.; New, L.; Moser, B.A.; Li, Y.; Liao, R.; Xie, C.; Chen, J.; Deng, Q.; Yamout, M.; et al. PRAK is essential for ras-induced senescence and tumor suppression. Cell 2007, 128, 295–308. [Google Scholar] [CrossRef] [PubMed]
  94. Choi, J.; Shendrik, I.; Peacocke, M.; Peehl, D.; Buttyan, R.; Ikeguchi, E.F.; Katz, A.E.; Benson, M.C. Expression of senescence-associated beta-galactosidase in enlarged prostates from men with benign prostatic hyperplasia. Urology 2000, 56, 160–166. [Google Scholar] [CrossRef]
  95. Ohuchida, K.; Mizumoto, K.; Murakami, M.; Qian, L.W.; Sato, N.; Nagai, E.; Matsumoto, K.; Nakamura, T.; Tanaka, M. Radiation to stromal fibroblasts increases invasiveness of pancreatic cancer cells through tumor–stromal interactions. Cancer Res. 2004, 64, 3215–3222. [Google Scholar] [CrossRef] [Green Version]
  96. Coppe, J.P.; Boysen, M.; Sun, C.H.; Wong, B.J.; Kang, M.K.; Park, N.H.; Desprez, P.Y.; Campisi, J.; Krtolica, A. A role for fibroblasts in mediating the effects of tobacco-induced epithelial cell growth and invasion. Mol. Cancer Res. 2008, 6, 1085–1098. [Google Scholar] [CrossRef] [Green Version]
  97. Székely, B.; Silber, A.L.; Pusztai, L. New Therapeutic Strategies for Triple-Negative Breast Cancer. Oncology 2017, 31, 130–137. [Google Scholar]
  98. Yoon, E.C.; Wilson, P.; Zuo, T.; Pinto, M.; Cole, K.; Harigopal, M. High frequency of p16 and SOX10 coexpression but not androgen receptor expression in triple-negative breast cancers. Hum. Pathol. 2020, 102, 13–22. [Google Scholar] [CrossRef]
  99. Bogina, G.S.; Lunardi, G.; Marcolini, L.; Brunelli, M.; Bortesi, L.; Marconi, M.; Coati, F.; Valerio, M.; Guerriero, M.; Massocco, A.; et al. P16 but not retinoblastoma expression is related to clinical outcome in no-special-type triple-negative breast carcinomas. Mod. Pathol. 2014, 27, 204–213. [Google Scholar] [CrossRef] [Green Version]
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Figure 1. Patients were divided into four groups according to the type of change in the breast: with IBC (n = 147), ISC (n = 79), AH (n = 82), and NE (n = 109). IHC expression of p16 was quantified in the nucleus and cytoplasm of tumor cells and presented as a percentage of positive in relation to the total number of evaluated cells. (A) The expression of p16 is significantly higher in IBC compared to NIL (p < 0.0005). The result is shown as the median. Statistical significance was analyzed using the Kruskal–Wallis and Mann–Whitney tests. (B–D) The increase in expression in IBC was accompanied by an increase in expression in ISC, AH, and NE. (E,F) Increased p16 expression in ISC was accompanied by increased expression in AH and NE. (G) Increased expression of p16 in AH is accompanied by an increase in expression in NE. Statistical significance was analyzed using the Spearman correlation coefficient. IHC expression of p16 in various lesions in the breast: (H) IBC. (I) ISC. (J) AH. (K) NE. (original magnification 200×).
Figure 1. Patients were divided into four groups according to the type of change in the breast: with IBC (n = 147), ISC (n = 79), AH (n = 82), and NE (n = 109). IHC expression of p16 was quantified in the nucleus and cytoplasm of tumor cells and presented as a percentage of positive in relation to the total number of evaluated cells. (A) The expression of p16 is significantly higher in IBC compared to NIL (p < 0.0005). The result is shown as the median. Statistical significance was analyzed using the Kruskal–Wallis and Mann–Whitney tests. (B–D) The increase in expression in IBC was accompanied by an increase in expression in ISC, AH, and NE. (E,F) Increased p16 expression in ISC was accompanied by increased expression in AH and NE. (G) Increased expression of p16 in AH is accompanied by an increase in expression in NE. Statistical significance was analyzed using the Spearman correlation coefficient. IHC expression of p16 in various lesions in the breast: (H) IBC. (I) ISC. (J) AH. (K) NE. (original magnification 200×).
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Figure 2. Expression of p16 in different molecular subtypes of breast cancer. All IBCs are divided into four molecular subtypes. IHC expression of p16 was quantified in the nucleus and cytoplasm of tumor cells and presented as a percentage of positive in relation to the total number of evaluated cells. (A) HER2+ subtype IBC showed the highest expression of p16. The result is shown as the median. Statistical significance was analyzed using the Kruskal–Wallis and Mann–Whitney tests (p = 0.025). (B) All IBCs were divided into negative and positive depending on HER2 status. There is no difference in p16 expression depending on HER2 expression. The result is shown as the median. Statistical significance was analyzed using the Mann–Whitney test (p = 0.293). (C) Ki67 expression is categorized into 3 groups: low (Ki67 < 15%), medium (Ki67: 16–30%) and high (Ki67 > 30%). The expression of p16 in tumor cells does not depend on the expression of Ki67. The result is shown as the median. Statistical significance was analyzed using the Kruskal–Wallis and Mann–Whitney tests (p = 0.123). (D,E) The ER and PR expression was analyzed through the Allred score. The increase in p16 expression in tumor cells was accompanied by significantly reduced ER and PR expression. Statistical significance was analyzed using by Spearman correlation coefficient (p = 0.011; r = −0.233; p = 0.007; r = −0.249). IHC expression of p16 in molecular subtypes—(F) Luminal A. (G) Luminal B. (H) HER2+. (I) TNBC (original magnification 200×).
Figure 2. Expression of p16 in different molecular subtypes of breast cancer. All IBCs are divided into four molecular subtypes. IHC expression of p16 was quantified in the nucleus and cytoplasm of tumor cells and presented as a percentage of positive in relation to the total number of evaluated cells. (A) HER2+ subtype IBC showed the highest expression of p16. The result is shown as the median. Statistical significance was analyzed using the Kruskal–Wallis and Mann–Whitney tests (p = 0.025). (B) All IBCs were divided into negative and positive depending on HER2 status. There is no difference in p16 expression depending on HER2 expression. The result is shown as the median. Statistical significance was analyzed using the Mann–Whitney test (p = 0.293). (C) Ki67 expression is categorized into 3 groups: low (Ki67 < 15%), medium (Ki67: 16–30%) and high (Ki67 > 30%). The expression of p16 in tumor cells does not depend on the expression of Ki67. The result is shown as the median. Statistical significance was analyzed using the Kruskal–Wallis and Mann–Whitney tests (p = 0.123). (D,E) The ER and PR expression was analyzed through the Allred score. The increase in p16 expression in tumor cells was accompanied by significantly reduced ER and PR expression. Statistical significance was analyzed using by Spearman correlation coefficient (p = 0.011; r = −0.233; p = 0.007; r = −0.249). IHC expression of p16 in molecular subtypes—(F) Luminal A. (G) Luminal B. (H) HER2+. (I) TNBC (original magnification 200×).
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Figure 3. ROC curve of p16 expression in NIL and IBC.
Figure 3. ROC curve of p16 expression in NIL and IBC.
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Figure 4. (A) p16 overexpression in stromal fibroblasts (original magnification 400×) and (B) rich mononuclear infiltrate in p16 positive IBCs (original magnification 400×).
Figure 4. (A) p16 overexpression in stromal fibroblasts (original magnification 400×) and (B) rich mononuclear infiltrate in p16 positive IBCs (original magnification 400×).
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Figure 5. p16 IHC, different subcellular localizations. (A) IBC—dominant cytoplasmic staining. (B) ISC—dominant cytoplasmic staining, with individual nuclear staining. (C) AH—dominant nuclear staining and individual cytoplasmic staining. (D) NE—nuclear staining (original magnification 200×).
Figure 5. p16 IHC, different subcellular localizations. (A) IBC—dominant cytoplasmic staining. (B) ISC—dominant cytoplasmic staining, with individual nuclear staining. (C) AH—dominant nuclear staining and individual cytoplasmic staining. (D) NE—nuclear staining (original magnification 200×).
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Table
Table 1. Clinicopathological characteristics of IBC.
Table 1. Clinicopathological characteristics of IBC.
VariablesN%
Sideleft6644.9
right8155.1
Histological typelobular1812.4
ductal12384.8
other42.8
Histological gradeHG11711.9
HG27351
HG35337.1
Nuclear gradeNG11715.2
NG26457.1
NG33127.7
Tumor necrosisabsent2621.7
present9478.3
Perineural invasionabsent10168.7
present4631.3
Lymphatic invasionabsent7248.9
present7551.1
Vascular invasionabsent11376.9
present3423.1
Molecular subtypesLuminal A3020.4
Luminal B7651.7
HER2+1912.9
TNBC2215
HER2negative11579.3
positive3020.7
Ki67low3020.9
medium4229.4
high7149.7
T statusT14835.8
T26447.8
T396.7
T4139.7
N statusN05037.3
N14835.8
N21914.2
N31712.7
Table
Table 2. Frequency of IBC molecular subtypes.
Table 2. Frequency of IBC molecular subtypes.
Molecular SubtypesN%
Luminal A3020.40
Luminal B7651.70
HER2+1912.93
TNBC2214.97
Total147100.0
Table
Table 3. Association between p16 expression in IBC and examined clinicopathological characteristics.
Table 3. Association between p16 expression in IBC and examined clinicopathological characteristics.
Variablesp16 Cut off 17.5Chi-SquaredfP
−+
Mononuclear infiltrateabsent2 (10%)2 (2.7%)7.95930.047
low13 (65%)30 (41.1%)
medium5 (25%)30 (41.1%)
high0 (0%)11 (15.1%)
Stromal fibroblastsabsent2 (9.1%)2 (3.2%)8.120 30.044
low7 (31.8%)32 (50.8%)
medium3 (13.6%)17 (27%)
high10 (45.5%)12 (19%)
Histological typelobular4 (13.8%)6 (6.7%)1.410 20.494
ductal24 (82.8%)80 (89.9%)
other1 (3.4%)3 (3.4%)
Histological gradeHG12 (8.3%)8 (10.5%)2.709 20.258
HG215 (62.5%)33 (43.4%)
HG37 (29.2%)35 (46.1%)
Nuclear gradeNG11 (4.8%)11 (14.8%)2.468 20.291
NG215 (71.4%)40 (54.1%)
NG35 (23.8%)23 (31.1%)
Tumor necrosis absent6 (23,1%)20 (21.3%)0.00011.000
present20 (76,9%)74 (78.7%)
Perineural invasionabsent18 (60%)65 (72.2%)1.05510.304
present12 (40%)25 (27.8%)
Lymphatic invasionabsent10 (33.3%)45 (50%)1.89110.169
present20 (66.7%)45 (50%)
Vascular invasionabsent23 (76.7%)66 (73.3%)0.01410.904
present7 (23.3%)24 (26.7%)
Molecular subtypesLuminal A4 (14.3%)11 (13.8%)3.029 30.387
Luminal B19 (67.9%)41 (51.2%)
HER2+2 (7.1%)11 (13.8%)
TNBC3 (10.7%)17 (21.2%)
HER2negative24 (82.8%)67 (75.3%)0.33410.563
positive5 (17.2%)22 (24.7%)
Ki67low4 (16.7%)11 (15.3%)7.217 20.027
medium11 (45.8%)14 (19.4%)
high9 (37.5%)47 (65.3%)
T statusT16 (22.2%)30 (37%)3.802 30.284
T214 (51.9%)40 (49.4%)
T33 (11.1%)3 (3.7%)
T44 (14.8%)8 (9.9%)
N statusN06 (33.3%)24 (40.7%)0.959 30.811
N19 (50%)22 (37.3%)
N22 (11.1%)8 (13.6%)
N31 (5.6%)5 (8.4%)
Table
Table 4. Frequency of p16 expression in different subcellular localizations of NIL and IBC epithelial cells.
Table 4. Frequency of p16 expression in different subcellular localizations of NIL and IBC epithelial cells.
p16 Nuclear vs. Cytoplasmic Expression
p16 IBC n (%)p16 ISC n (%)p16 AH n (%)p16 NE n (%)
negative 3 (2.3)3 (3.8)3 (3.7)3 (2.8)
nuclear28 (21.1)20 (25.6)38 (46.3)99 (90.8)
cytoplasmic102 (76.6)55 (70.6)41 (50)7 (6.4)
Total 133 (100)78 (100)82 (100)109 (100)
Table
Table 5. Association of subcellular localization of p16 protein with clinicopathological characteristics of IBC.
Table 5. Association of subcellular localization of p16 protein with clinicopathological characteristics of IBC.
Variablesp16 Expression in IBCChi-SquaredfP
NuclearCytoplasmic
Mononuclear infiltrateabsent1 (5%)2 (2.9%)16.236 60.013
low14 (70%)26 (37.7%)
medium5 (25%)30 (43.5%)
high0 (0%)11 (15.9%)
Stromal fibroblastsabsent1 (5%)2 (3.2%)11.638 60.071
low8 (40%)31 (50%)
medium6 (30%)13 (21%)
high5 (25%)16 (25.8%)
Tumor necrosisabsent7 (58.3%)19 (17.6%)6.609 20.037
present5 (41.7%)89 (82.4%)
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Jovanovic, D.V.; Mitrovic, S.L.; Milosavljevic, M.Z.; Ilic, M.B.; Stankovic, V.D.; Vuletic, M.S.; Dimitrijevic Stojanovic, M.N.; Milosev, D.B.; Azanjac, G.L.; Nedeljkovic, V.M.; et al. Breast Cancer and p16: Role in Proliferation, Malignant Transformation and Progression. Healthcare 2021, 9, 1240. https://doi.org/10.3390/healthcare9091240

AMA Style

Jovanovic DV, Mitrovic SL, Milosavljevic MZ, Ilic MB, Stankovic VD, Vuletic MS, Dimitrijevic Stojanovic MN, Milosev DB, Azanjac GL, Nedeljkovic VM, et al. Breast Cancer and p16: Role in Proliferation, Malignant Transformation and Progression. Healthcare. 2021; 9(9):1240. https://doi.org/10.3390/healthcare9091240

Chicago/Turabian Style

Jovanovic, Dalibor V., Slobodanka L. Mitrovic, Milos Z. Milosavljevic, Milena B. Ilic, Vesna D. Stankovic, Milena S. Vuletic, Milica N. Dimitrijevic Stojanovic, Danijela B. Milosev, Goran L. Azanjac, Vladica M. Nedeljkovic, and et al. 2021. "Breast Cancer and p16: Role in Proliferation, Malignant Transformation and Progression" Healthcare 9, no. 9: 1240. https://doi.org/10.3390/healthcare9091240

APA Style

Jovanovic, D. V., Mitrovic, S. L., Milosavljevic, M. Z., Ilic, M. B., Stankovic, V. D., Vuletic, M. S., Dimitrijevic Stojanovic, M. N., Milosev, D. B., Azanjac, G. L., Nedeljkovic, V. M., & Radovanovic, D. (2021). Breast Cancer and p16: Role in Proliferation, Malignant Transformation and Progression. Healthcare, 9(9), 1240. https://doi.org/10.3390/healthcare9091240

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