Therapy-induced senescent cancer cells exhibit complement activation and increased complement regulatory protein expression.

Therapy-induced senescence (TIS) is a primary response to chemotherapy, contributing to untoward treatment outcomes such as evasion of immunosurveillance. Despite the established role of the complement system in the immune response to cancer, the role of complement in mediating the immune response against senescent tumor cells remains poorly understood. To explore this relationship, we exposed lung adenocarcinoma (A549), breast adenocarcinoma (MCF7) and pancreatic carcinoma (Panc-1) cell lines to sublethal doses of either etoposide or doxorubicin to trigger TIS. Identification of TIS was based on morphological changes, upregulation of the senescence-associated ß-galactosidase, p21Cip1 induction and lamin B1 downregulation. Using immunofluorescence microscopy, quantitative PCR, ELISA of conditioned media and in silico analysis, we investigated complement activation, complement protein expression, C3 levels in the conditioned media of senescent cells and secreted complement proteins as part of the senescence-associated secretory phenotype (SASP), respectively. In cell lines undergoing TIS, complement-related changes included (i) activation of the terminal pathway, evidenced by the deposition of C5b-9 on senescent cells; (ii) an increase in the expression of CD59 and complement factor H and (iii) in A549 cells, an elevation in the expression of C3 with its secretion into the medium. In addition, increased C3 expression was observed in breast cancer samples expressing TIS hallmarks following exposure to neoadjuvant chemotherapy. In conclusion, TIS led to the activation of complement, upregulation of complement regulatory proteins and increased C3 expression. Complement appears to play a role in shaping the cancer microenvironment upon senescence induction.

Characterization of BCL-X L , MCL-1, and BAX Protein Expression in Response to Neoadjuvant Chemotherapy in Breast Cancer.

The use of chemotherapy has improved the overall treatment of breast cancer, which is frequently administered in the form of neoadjuvant chemotherapy (NAC). Apoptosis is an established cell stress response to NAC in preclinical models; however, there is limited understanding of its role in clinical cancer, specifically, its contribution to favorable pathologic responses in breast cancer therapy. Here, we aimed to characterize the change in protein expression of 3 apoptosis-associated biomarkers, namely, BCL-X L , MCL-1, and BAX in breast cancer in response to NAC. For this, we utilized a set of 68 matched invasive breast cancer FFPE samples that were collected before (pre) and after (post) the exposure to NAC therapy that were characterized by incomplete pathologic response. Immunohistochemistry (IHC) analysis suggested that most of the samples show a decrease in the protein expression of all 3 markers following exposure to NAC as 90%, 69%, and 76% of the matched samples exhibited a decrease in expression for BCL-X L , MCL-1, and BAX, respectively. The median H-score of BCL-X L post-NAC was 150/300 compared with 225/300 pre-NAC ( P value <0.0001). The median H-score of MCL-1 declined from 200 pre-NAC to 160 post-NAC ( P value <0.0001). The median H-score of BAX protein expression decreased from 260 pre-NAC to 190 post-NAC ( P value <0.0001). There was no statistically significant association between the expression of these markers and stage, grade, and hormone receptor profiling (luminal status). Collectively, our data indicate that the expression of apoptosis regulatory proteins changes following exposure to NAC in breast cancer tissue, developing a partial pathologic response.

A three-marker signature identifies senescence in human breast cancer exposed to neoadjuvant chemotherapy.

PURPOSE: Despite the beneficial effects of chemotherapy, therapy-induced senescence (TIS) manifests itself as an undesirable byproduct. Preclinical evidence suggests that tumor cells undergoing TIS can re-emerge as more aggressive divergents and contribute to recurrence, and thus, senolytics were proposed as adjuvant treatment to eliminate senescent tumor cells. However, the identification of TIS in clinical samples is essential for the optimal use of senolytics in cancer therapy. In this study, we aimed to detect and quantify TIS using matched breast cancer samples collected pre- and post-exposure to neoadjuvant chemotherapy (NAC). METHODS: Detection of TIS was based on the change in gene and protein expression levels of three senescence-associated markers (downregulation of Lamin B1 and Ki-67 and upregulation of p16INK4a). RESULTS: Our analysis revealed that 23 of 72 (31%) of tumors had a shift in the protein expression of the three markers after exposure to NAC suggestive of TIS. Gene expression sets of two independent NAC-treated breast cancer samples showed consistent changes in the expression levels of LMNB1, MKI67 and CDKN2A. CONCLUSIONS: Collectively, our study shows a more individualized approach to measure TIS hallmarks in matched breast cancer samples and provides an estimation of the extent of TIS in breast cancer clinically. Results from this work should be complemented with more comprehensive identification approaches of TIS in clinical samples in order to adopt a more careful implementation of senolytics in cancer treatment.

NOXA expression is downregulated in human breast cancer undergoing incomplete pathological response and senescence after neoadjuvant chemotherapy.

Neoadjuvant chemotherapy (NAC) is a frequently utilized approach to treat locally advanced breast cancer, but, unfortunately, a subset of tumors fails to undergo complete pathological response. Apoptosis and therapy-induced senescence (TIS) are both cell stress mechanisms but their exact role in mediating the pathological response to NAC is not fully elucidated. We investigated the change in expression of PAMIP1, the gene encoding for the pro-apoptotic protein, NOXA, following NAC in two breast cancer gene datasets, and the change in NOXA protein expression in response to NAC in 55 matched patient samples (pre- and post-NAC). PAMIP1 expression significantly declined in post-NAC in the two sets, and in our cohort, 75% of the samples exhibited a downregulation in NOXA post-NAC. Matched samples that showed a decline in NOXA post-NAC were examined for TIS based on a signature of downregulated expression of Lamin-B1 and Ki-67 and increased p16INK4a, and the majority exhibited a decrease in Lamin B1 (66%) and Ki-67 (80%), and increased p16INK4a (49%). Since our cohort consisted of patients that did not develop complete pathological response, such findings have clinical implications on the role of TIS and NOXA downregulation in mediating suboptimal responses to the currently established NAC.

PD-L1 expression in breast invasive ductal carcinoma with incomplete pathological response to neoadjuvant chemotherapy.

OBJECTIVES: To investigate the expression of programmed death-ligand 1 (PD-L1) in breast cancer in association with incomplete pathological response (PR) to neoadjuvant chemotherapy (NAC). METHODS: PD-L1 expression was evaluated using immunohistochemistry in post-operative, post-NAC samples of 60 patients (n = 60) diagnosed with breast invasive ductal carcinoma with incomplete PR to NAC, including 31 matched pre-NAC and post-NAC samples (n = 31). PD-L1 protein expression was assessed using three scoring approaches, including the tumor proportion score (TPS), the immune cell score (ICS), and the combined tumor and immune cell score (combined positive score, CPS) with a 1% cut-off. RESULTS: In the post-operative, post-NAC samples (n = 60), positive expression rate of PD-L1 was observed in 18.3% (11/60) of cases by TPS, 31.7% (19/60) by ICS, and 25% (15/60) by CPS. In matched samples, positive expression rate of PD-L1 was observed in 19.3% (6/31) of patients by TPS, 51.6% (16/31) by ICS, and 19.3% (6/31) by CPS in pre-NAC specimens, while it was observed in 22.6% (7/31) of matched post-NAC samples by TPS, 22.6% (7/31) by ICS, and 19.3% (6/31) by CPS. In the matched samples, there was a significant decrease in PD-L1 immunoexpression using ICS in post-NAC specimens (McNemar's, p = 0.020), while no significant differences were found using TPS and CPS between pre- and post-NAC samples (p = 1.000, p = 0.617; respectively). PD-L1 immunoexpression determined by TPS or CPS was only significantly associated with ER status (p = 0.022, p = 0.021; respectively), but not with other clinicopathological variables. We could not establish a correlation between PD-L1 expression and the overall survival rate (p > 0.05). There were no significant differences in the tumor infiltrating lymphocytes count between the paired pre- and post-NAC samples (t = 0.581, p = 0.563 or Wilcoxon's Signed Rank test; z = -0.625, p = 0.529). CONCLUSION: Our findings indicate that PD-L1 protein expression in infiltrating immune cells was significantly reduced in breast tumors that developed incomplete PR following the exposure to NAC.

The Expression of the Senescence-Associated Biomarker Lamin B1 in Human Breast Cancer.

Senescence is a major response to cancer chemotherapy and has been linked to unfavorable therapy outcomes. Lamin B1 is a component of the nuclear lamina that plays a pivotal role in chromatin stability. Downregulation of lamin B1 represents an established biomarker for cellular senescence. However, the protein expression level of lamin B1 in malignant tissue, particularly of the breast, has not been previously described. In this work, we investigated lamin B1 protein expression in normal breast epithelium, malignant breast tissue (including adjacent non-malignant tissue) and in malignant tissue exposed to neoadjuvant chemotherapy (NAC) using immunohistochemistry (IHC) in three patient groups (n = 15, n = 87, and n = 43, respectively). Our results indicate that lamin B1 mean positive expression was 93% in normal breast epithelium and 88% in malignant breast cells, but significantly decreased (mean: 55%, p < 0.001) in malignant breast tissue after exposure to NAC, suggestive of senescence induction. No significant association between lamin B1 expression and other clinicopathological characteristics or survival of breast cancer patients was recorded. To our knowledge, this is the first report that established the baseline protein expression level of lamin B1 in normal and malignant breast tissue, and its reduction following exposure to chemotherapy. In conclusion, lamin B1 downregulation can be used reliably as a component of multiple biomarker batteries to identify therapy-induced senescence (TIS) in clinical cancer.

Expression of Senescence and Apoptosis Biomarkers in Synchronous Bilateral Breast Cancer: A Case Report.

BACKGROUND: Synchronous bilateral breast cancer (SBBC) provides a special condition where two independent breast tumors are exposed to cancer pharmacotherapy within a uniform pharmacokinetic milieu. Both senescence and apoptosis are established responses to therapy; however, they have potentially variable contributions to the overall outcome of treatment, which are yet to be determined. METHODS: In this report, we describe the clinicopathological picture of two SBBC cases that received standard anticancer treatment and assess their expression profile of several molecular hallmarks of senescence and apoptosis. RESULTS: Our analysis identified that synchronous tumors have variable expression profiles of both senescence- and apoptosis-associated biomarkers, despite comparable pathological responses to neoadjuvant chemotherapy and current survival rates. CONCLUSIONS: Our results highlight the variable expression of senescence- and apoptosis-associated markers in breast tumors (despite the shared somatic genetic background) and invites a large-scale assessment of both senescence and apoptosis in breast cancer tissue in vivo and their contribution to the pathological response and overall survival.

An Uncommon Recurrent Metastasis of Ovarian Immature Teratoma to the Small Bowel.

Immature ovarian teratomas are rare ovarian germ cell tumors associated with a variable potential of distant metastasis. The acquisition of mature phenotypes upon post-treatment recurrence of immature teratomas has been previously described. In this study, we report, for the first time, a rare case of a recurrent ovarian immature teratoma with mature deposits in the small bowel. An incidental pelvi-abdominal mass was identified in a 30-year-old pregnant patient during antenatal ultrasonography. The mass, which was resected through salpingo-oopherectomy, was histopathologically characterized as an immature teratoma of grade 2 and treated with 3 cycles of chemotherapy. After 3 years of completing treatment, the patient suffered from severe anemia which was investigated by capsule endoscopy that identified a bleeding source in the ileum. Imaging studies revealed an intrabdominal mass that was resected laparoscopically. The pathological assessment of the mass identified a submucosal/intramuscular teratoma with mature elements indicative of a recurrent metastasis of immature teratoma associated with post-chemotherapy retroconversion. The secondary mass was then managed with adjuvant chemotherapy.

Expression of therapy-induced senescence markers in breast cancer samples upon incomplete response to neoadjuvant chemotherapy.

Senescence is a cell stress response induced by replicative, oxidative, oncogenic, and genotoxic stresses. Tumor cells undergo senescence in response to several cancer therapeutics in vitro (Therapy-Induced Senescence, TIS), including agents utilized as neoadjuvant chemotherapy (NAC) in the treatment of invasive breast cancer. TIS has been proposed to contribute to adverse therapy outcomes including relapse. However, there is limited evidence on the induction of senescence in response to NAC in clinical cancer and its contribution to disease outcomes. In this work, the expression of three senescence-associated markers (p21CIP1, H3K9Me3 (histone H3 lysine 9 trimethylation), and Lamin B1) was investigated in breast cancer samples that developed partial or incomplete pathological response to NAC (n=37). Accordingly, 40.54% of all samples showed marker expression consistent with a senescence-like phenotype, while the remainders were either negative or inconclusive for senescence (2.70 and 56.8%, respectively). Moreover, analysis of core-needle biopsies revealed minimal changes in p21CIP1 and H3K9Me3, but significant changes in Lamin B1 expression levels following NAC, highlighting a more predictive role of Lamin B1 in senescence detection. However, our analysis did not establish an association between TIS and cancer relapse as only three patients (8.1%) with a senescence-like profile developed short-term recurrent disease. Our analysis indicates that identification of TIS in tumor samples requires large-scale transcriptomic and protein marker analyses and extended clinical follow-up. Better understanding of in vivo senescence should elucidate its contribution to therapy outcomes and pave the way for the utilization of senolytic approaches as potential adjuvant cancer therapy.