1. Introduction
Hallmark characteristics of cellular senescence include withdrawal from the cell cycle, macromolecular damage, deregulated metabolism, and the production of a senescence-associated secretory phenotype (SASP), in which a variety of factors are secreted that can influence the behavior of neighboring and immune cells [
1,
2,
3,
4,
5,
6]. The induction of senescence as a consequence of oncogene activation is considered to represent a barrier to tumorigenesis [
1,
7]. Consistently, senescent cells have been observed within premalignant tumors [
8,
9,
10,
11,
12,
13]. For cancer development, it has therefore been suggested that the precursors of tumor cells need to circumvent or escape from oncogene-induced senescence and gain the ability to proliferate while expressing activated oncogenes [
14].
The tumor suppressor signaling pathways Arf-p53 and pRB-16INK4a facilitate the induction of senescence in response to oncogenic stimuli [
1,
15]. Evasion of senescence and escape from growth arrest is fostered by cell-autonomous mechanisms that involve genetic or epigenetic changes. As an example, functional defects in Arf-p53 and pRB-p16INK4a signaling can prevent the induction of senescence and promote malignant progression [
12,
16,
17,
18]. Furthermore, tumor-infiltrating immune cells can counter senescence through non-cell autonomous mechanisms that protect proliferating tumor cells from senescence or enable already arrested cells to escape from oncogene-induced senescence, thus sustaining tumor growth [
19].
Senescence can be induced in tumor cells [
7], suggesting that senescence is more than just a hurdle to tumorigenesis during the development of cancer. For example, conventional cancer therapies can induce senescence in tumor cells [
20]. Mechanistically this can be due to p53 reactivation [
21,
22] or loss of Skp2 [
23] in tumor cells or can be caused by T-helper cells that invade the tumor and secrete cytokines such as IFN-γ and tumor necrosis factor (TNF) that stimulate senescence [
24]. The induction of senescence can lead to regression of the tumor and may be coupled with an inflammatory response that induces immune cells to destroy and clear the senescent tumor cells [
2,
22,
25].
Paradoxically, senescent cells within tumors can promote the growth and progression of non-senescent cancer cells. Many SASP components are pro-tumorigenic growth factors, matrix metalloproteinases (MMPs), and cytokines that are known to stimulate the aggressive behavior of cancer cells in vitro [
26,
27,
28,
29,
30]. Through their SASP, senescent cells can promote the progression of both precancerous cells and established cancer cells in mouse xenograft models [
26,
31]. For example, when breast cancer cells that express constitutively active HER2 enter senescence, the SASP they produce stimulates the metastatic progression of non-senescent tumor cells and inhibits the immune clearance of senescent cells [
32]. In B-Raf mutated papillary thyroid carcinomas, senescent cancer cells promote collective invasion of senescent and non-senescent cancer cells and foster metastatic progression through their SASP [
30]. In colorectal cancers, senescent tumor cells protect non-senescent cancer cells from immune cells by building a cytokine shield out of SASP components [
6]. In the context of prostate cancer, loss of tissue inhibitor of metalloproteinases-1 (TIMP1) causes activation of MMPs and thereby reprogramming of the SASP into a SASP that fosters metastasis [
33]. MMPs in the SASP can also cleave NKG2D ligands, which suppresses NK cell-dependent immune surveillance and enables senescent cells to evade immune clearance [
34]. The latter examples illustrate the particular importance of MMPs, which are major components of SASPs, in promoting the progression of malignancies by either facilitating migration and invasion or enabling cancer cells to evade immune clearance.
Although SASP components may promote malignant progression through paracrine signaling, the composition of SASPs is complex, and not all SASP factors promote cancerogenesis [
1], and some SASP components stimulate the elimination of senescent cells by the immune system [
2,
5,
22]. Thus, the recruitment of immune cells and the consequences of the immune response to senescent cells is determined by the exact composition of the SASP. Furthermore, the effect of individual SASP components depends on the cellular microenvironment and tissue-specific context [
5,
32]. Moreover, senescence is not always irreversible. Molecular mechanisms exist that enable tumor cells to escape from senescence and cancer cells that exit senescence acquire a more aggressive phenotype [
35].
The ability of senescence to act as a barrier to tumor progression on the one hand but also to promote tumor initiation and metastasis on the other has been demonstrated in murine xenograft and transgenic models [
2,
22,
27,
30,
31,
32,
33,
34,
35,
36,
37]. Senescent tumor cells have been observed within several therapy-naïve human carcinomas [
30,
38]. In human therapy-naïve breast carcinomas, senescent tumor cells exist, and their presence is dependent on the molecular subtype [
38]. However, the possible existence of senescent tumor cells within breast cancer metastases has not been explored to date. In the present study, we investigated senescence-associated β-galactosidase (SA-β-gal) activity within primary luminal breast cancer samples and their matched sentinel lymph node metastases from patients who had not been treated with neoadjuvant therapy or radiotherapy. We also investigated whether senescent tumor cells exist within metastatic lesions from patients with other breast cancer subtypes.
We found similarities in the appearance of senescent cancer cells between primary tumors and their corresponding metastases within luminal A and B breast carcinomas. Analysis of lymph nodes from patients with other breast cancer subtypes also revealed senescent tumor cells within metastatic lesions. As senescent cancer cells avoid recognition by the immune system through MMPs in their SASP [
34], these observations might reflect the ineffective elimination of senescent breast cancer cells by the immune system due to SASP-mediated suppression of immune surveillance and might also explain why breast carcinomas show an inefficient response to cancer immunotherapies. Taken together, our observations raise the question as to whether the targeting of senescent tumor cells with senolytic or senomorphic drugs might be beneficial for improving the treatment of breast and other cancers.
4. Discussion
Cellular senescence is considered to be a barrier to oncogene-induced carcinogenesis [
1,
20]. A few early studies reported senescent cells in premalignant human naevi and colon adenomas, but in comparison, no or significantly reduced numbers of senescent cells were found in malignant melanomas and adenocarcinomas. These studies concluded that cellular senescence might not be relevant in the context of advanced cancers and their metastases [
8,
9,
10,
11,
12,
13]. However, later studies clearly showed that in the context of untreated invasive human carcinomas from colon and BRAF-mutated thyroid cancer patients, significant numbers of senescent cells could be present in the primary tumor [
6,
30]. We have previously reported that senescent tumor cells also exist within human breast tumors [
38]. To follow up on this finding, in the present study, we evaluated the presence of senescent cancer cells within primary invasive breast carcinomas and their matched SLN metastases. Our results show that the majority of the primary luminal tumors and their matched metastases contain large numbers of senescent cells, which was also true for SLNs taken from pre-therapy patients with HER2+ and TNBC breast cancers. These observations are consistent with our previous work in which we observed high numbers of SA-β-gal positive tumor cells within HER2+ primary tumors [
38]. Taken together, our findings suggest that cellular senescence is a feature of metastases from all breast cancer molecular subtypes.
Senescent tumor cells can contribute to metastatic dissemination in a number of ways, including the fostering of tumor cell invasion [
29] and metastatic progression [
30] and protecting against anti-tumor immune responses [
6]. Mechanistically, the SASP produced by senescent cells in cancer tissue plays a major role in increasing the malignant potency of cancer cells [
31]. For example, cancer therapies can induce senescence in both the tumor microenvironment and in cancer cells, and the SASPs that are produced as a consequence can negatively impact treatment efficacy in a number of ways and cause cancer progression [
45]. Consistently, the targeting of therapy-induced senescent cells by senolytics or by suppressing the secretion of SASPs has been shown to improve cancer treatment outcomes [
46]. For example, in ovarian cancer, platinum-containing chemotherapy induces cellular senescence and an associated SASP that promotes cancer stem cell formation and promotes tumor recurrence. This can be suppressed by treatment with the NAMPT inhibitor FK866 [
47]. In prostate cancer, radiation treatment leads to the induction of senescence in prostate cancer cells and fibroblasts and the release of an NFkB-driven pro-inflammatory SASP. The mTOR-inhibitor rapamycin prevents the expression of the SASP induced by radiation treatment and thereby inhibits tumor progression [
48,
49]. These examples illustrate that targeting therapy-induced senescent cells can improve cancer treatment.
In BRAF-mutated papillary thyroid carcinomas, senescent tumor cells promote the collective invasion of senescent and non-senescent tumor cells via their SASP. Furthermore, the senescent tumor cells induce anoikis resistance in non-senescent tumor cells during the passage through the blood and lymph flow, helping non-senescent tumor cells to survive within the circulatory system [
30]. Collective invasion is a feature of the majority of human breast carcinomas [
50]. Thus, it is conceivable that senescent cells also support the collective invasion and the metastatic process in breast carcinomas through their SASP in a manner similar to that observed in papillary thyroid carcinomas, in particular in breast cancer subtypes with a high incidence of senescent tumor cells such as luminal A and HER2+ [
38]. Furthermore, cell cycle-arrested senescent cells are likely to be less susceptible to anti-cancer drugs and are not recognized by the immune system. They might therefore be able to remain dormant for prolonged periods but eventually regain proliferative capacity and form metastases [
51].
Interestingly, the SASP of senescent cells transformed by constitutive HER2 signaling inhibits the clearance of senescent cells and exerts pro-metastatic effects leading to breast cancer progression in mice xenografts [
32]. Taken together, these observations suggest that further investigations should focus on defining the constitution of the SASP produced by senescent cells in human luminal A and HER2+ breast carcinomas and determining how different SASP components foster cell survival, anoikis resistance, dormancy, drug resistance, and protection from the anti-tumor immune response.
Luminal A breast carcinomas are well differentiated, have a low Ki67 index, and are associated with a good prognosis. However, these tumors are characterized by late recurrence and the formation of metastases many years after apparently successful treatment. Thus, these tumors seem to disseminate early, and tumor cells stay dormant over a long period of time. The dormant properties of these cells may be linked to senescence. Cancer cells can exit senescence through cell and non-cell autonomous mechanisms such as alterations within the tumor suppressor pathways p53-p21
Cip1/Waf1 and pRB-p16
INK4A or through the infiltration of immune cells, such as myeloid cells, that enable already arrested cells to escape from oncogene-induced senescence, and thus sustain tumor growth [
19]. Following escape from therapy-induced senescence, tumor cells acquire stem-like properties and a more aggressive phenotype, which is paralleled in breast cancer patients with recurrent tumors [
35].
Recurrence within luminal breast cancer patients is strongly associated with resistance to endocrine treatment [
52]. SASP components produced by senescent luminal breast cancer cells could conceivably lead to the activation of signaling pathways such as Wnt and STAT3 that are associated with resistance to endocrine treatment [
53,
54], or promote the survival of disseminated tumor cells in the presence of endocrine treatment over long periods of time. Further studies are required to understand the role of senescent luminal A tumor cells and their SASPs in resistance to endocrine therapies and whether targeting senescent luminal breast carcinoma cells might be useful to overcome endocrine resistance and avoid late recurrence.
Previously we have reported that few, if any, SA-β-gal positive tumor cells exist within primary human TNBCs [
38]. Surprisingly, we observed SA-β-gal positive tumor cells within all of the TNBC metastases analyzed. A possible explanation for the high numbers of SA-β-gal positive tumor cells within SLN metastases from TNBC patients might be that these tumor cells have a higher mutational burden and might therefore be better detected by the immune system and thus more accessible to immune surveillance. Tumor-infiltrating T-helper 1 cells can induce senescence in tumor cells through secretion of the cytokines IFN-γ and tumor necrosis factor (TNF) [
24]. Thus, it is conceivable that senescence is induced in TNBC cells when they encounter CD4+ T-cells in the SLNs. It can be speculated that these tumor cells must activate escape mechanisms to leave the LNs and to form metastases at distant organ sides. Escape from therapy-induced senescence is associated with a stem-like phenotype and more aggressive behavior [
35]. Maybe this mechanism also represents a selection process within the LNs for immune evasion and escape from immune cell-induced senescence ending up in aggressive, stem-like TNBC cells. Interestingly, certain tumor-infiltrating immune cells can oppose senescence in murine models [
19]. It was shown that CD11b(+)Gr-1(+) myeloid cells can protect proliferating tumor cells from senescence or enable already arrested cells to escape from oncogene-induced senescence by antagonizing senescence in a paracrine manner through interfering with the SASP of the tumor and secretion of interleukin-1 receptor antagonist (IL-1RA) [
19]. Possibly, myeloid-derived suppressor cells might be involved in the escape from immune cell-induced senescence and, as a consequence, in metastatic progression.
A limitation of this study is the relatively low number of SLN metastases analyzed, as well as the fact that we could only make a direct comparison between primary tumors and SLN metastases in the context of luminal A and B breast cancers. The widespread neoadjuvant treatment of breast cancer patients strongly restricts the possibility of collecting untreated primary tumors and their matched metastases. Nevertheless, it is notable that significantly fewer ILC SLN samples contained senescent cells compared to SLN from IDC, despite the relatively low number of ILC samples in this analysis (
Table 6). Although IDCs and ILCs are histologically and clinically different, ILC is nevertheless treated similarly to IDC [
55]. However, ILC is less responsive to systemic chemotherapy compared to IDC [
55]. Differences in the number of SA-β-gal positive tumor cells between ILC and IDC metastases further highlight differences between these two histologic BC subtypes. In ILC,
CDH1,
PIK3CA,
TBX3,
FOXA1, and
RUNX1 are the most commonly mutated genes [
56]. Remarkably, genetic alterations in
PIK3CA,
TBX3,
FOXA1, and
RUNX1 are associated with escape from oncogene-induced senescence and resistance to senescence [
57,
58,
59,
60,
61] and systemic chemotherapy [
62,
63]. It is, therefore, tempting to speculate that the trend for ILC SLN metastases to contain fewer senescent cells compared to IDC SLN metastases (
Table 6) may reflect the fact that ILC harbors more defects in genes that help cells to escape from senescence or to prevent oncogene-induced senescence.
In the context of aging, the extent to which the immune system is involved in regulating the number of senescent cells and whether age-related impairment of immune function contributes to the accumulation of senescent cells in the elderly is unknown [
25]. Theoretically, the accumulation of senescent cells in breast tissue due to either aging or impaired immune surveillance could promote the development of breast cancer and later its progression. However, we did not observe an accumulation of senescent cells in the normal breast tissue surrounding tumors, even in patients at older ages (
Table 4 and
Figure 5). As we observed senescent tumor cells only within tumor tissue, we speculate that this reflects the ability of tumor cells to evade immune clearance. Recently, for example, it was shown that senescent cells foster the avoidance of immune recognition through MMPs in their SASP through a mechanism that involves MMP-dependent shedding of NKG2D-ligands that results in the suppression of NKG2D-mediated immunosurveillance [
34]. Consistent with this notion, most breast carcinomas respond only inefficiently towards cancer immunotherapies, and a potential explanation might be that senescent breast cancer cannot be cleared by the immune system due to SASP-mediated suppression of immune surveillance. Thus, targeting senescent breast cancer cells with either senolytic or senomorphic drugs might render breast cancer more susceptible to the immune system and towards anti-cancer therapies.
In summary, this study demonstrates for the first time that senescent tumor cells exist within advanced human primary luminal breast carcinomas and that senescent tumor cells also exist within their matched lymph node metastasis. Several reports based on murine xenografts point to a metastasis-promoting function of senescent tumor cells. Since luminal breast carcinomas are characterized by late recurrence and poor susceptibility to chemotherapy and anti-tumor immunotherapies, it will be of importance to understand whether a correlation exists between senescence and tumor dormancy and whether senescence participates in tumor immune evasion. Further investigations focusing on the functional characterization of senescent breast cancer cells and their SASPs will be of importance to clarify the degree to which such senescent cells and their SASPs contribute to metastatic progression. Translationally, the targeting of senescent cancer cells or inhibiting the activity of SASP factors produced by senescent cancer cells holds promise as a strategy to improve breast cancer treatment and avoid late recurrence. It is of note that senolytic drugs such as ABT-263 induce apoptosis, leading to the clearance of therapy-induced senescent tumor cells [
64]. Thus, it will be of particular interest to determine whether senolytic drugs can clear senescent breast cancer cells from breast carcinomas and thereby suppress therapy resistance, recurrence, and metastasis.