3.1 RPL32 expression in pre-neoadjuvant core needle biopsies of aggressive breast cancer
IHC was used to study the expression of RPL32 in TNBC tissues. The RPL32 protein in these tissues was located in the cytoplasm. There was a variable expression among the 96 breast cancer tissues, 30 (31.25%) were being positive(Fig. 1A), 66 (68.75%) were negative(Fig. 1B) for RPL32 protein. Whereas, no expression was observed in normal 22 samples of breast tissue(Fig. 1C).
3.2 Relationship between RPL32 expression and clinical features in TNBC patients
The RPL32 expression in TNBC tissues showed no assoscation with patient age, tumor grade, tumor size, clinical staging, ECOG, and Ki67 expression (p = .124, p = .612, p = .447, p =.156, p = .137, p = .372 , respectively) . Whereas, the expression of RPL32 was found to be correlated with MP grading, lymph node metastasis, and whether achieved pCR (p = .029, p = .027, p = .033) (Table 1).
3.3 RPL32 upregulation promotes adriamycin chemoresistance in TNBC cells
To test the hypothesis, TNBC cell lines MDA-MB-231 and TB549 that stably express RPL32 were constructed. RPL32 levels were considerably higher in MDA-MB-231 and TB549 cell lines, according to western blot analysis as shown in Figure 2A. Upregulation of RPL32 significantly improved the capacity of TNBC cell lines MDA-MB-231 and TB549 to form colonies (Figure 2B). Furthermore, in RPL32 overexpressing cells, the half-maximum inhibitory concentration (IC50) of adriamycin was considerably increased (Figure 2 C-D). In RPL32 overexpressing cells treated with Adriamycin, FACS analysis revealed reduced apoptotic rates (Figure 2E). According to our findings, the RPL32 has a critical role in adriamycin chemoresistance in TNBC.
3.4 Silencing of RPL32 induces adriamycin chemosensitivity in TNBC cells.
RPL32-silenced stable cell lines (MDA-MB-231 and TB549) were developed to further investigate the role of RPL32 in the development of TNBC cell chemoresistance (Figure 3A). Downregulation of RPL32 remarkably decreased the colony formation ability of MDA-MB-231 and TB549 cell lines (Figure 3B). In TNBC cell lines, downregulation of RPL32 reduced the IC50 value of adriamycin (Figure 3C-D). Furthermore, following adriamycin treatment, the apoptosis in RPL32-knockdown cells was greater than in vector control cells (Figure 3E). Finally, RPL32 knockdown improves TNBC cancer cells' chemosensitivity to adriamycin therapy.
3.5 RPL32 activates the Akt signaling pathway in TNBC cells.
The western blot analysis showed that RPL32 knockdown increased the apoptosis, as shown by higher expression of cleaved caspase-3 and Bax proteins, and a decrease in Bcl2 expression, while RPL32 overexpression induced the opposite effects (Figure 4A). In different cancer types, constitutive activation of the Akt signaling pathway provides chemoresistance[20, 21]. Immunoblotting analysis was used to assess the influence of RPL32 on the activation of the Akt signaling pathway to determine if RPL32 enhances chemoresistance through regulation of the Akt pathway. RPL32 overexpression enhanced (but RPL32 knockdown suppressed) Akt phosphorylation, as revealed by our findings (Figure 4B). RPL32-upregulated cells were treated with a combination of adriamycin and vehicle, or Adriamycin with the Akt inhibitor MK-2206, to see whether RPL32-induced chemoresistance was reliant on the Akt pathway. Our findings showed that MK-2206 abolished RPL32 overexpression-repressed apoptotic level in TNBC cells (Figure 4C), signifying that Akt inhibitor renders the sensitivity of TNBC cells to adriamycin therapy.
3.6 High expression of RPL32 is related to poor prognosis of neoadjuvant chemotherapy in TNBC
The survival status of 96 patients with TNBC who had neoadjuvant treatment was analyzed using the Kaplan-Meier method. The findings showed that Age, tumor grade and size, clinical stage, ECOG score, and Ki-67 index had little effect on the prognosis of patients, while lymph node metastasis, MP grade, and patient's response to chemotherapy were correlated with their prognosis. High RPL32 expression was related to a lower DFS (median DFS, 44 months vs 33 months, p = .041), as well as a lower DDFS (median DDFS, 47 months vs 36 months, p = .044). Similarly, High expression of RPL32 was significantly correlated with lower OS (median OS, 52 months vs 40 months, p = .041). These findings suggest that increased RPL32 expression is correlated to a poor prognosis in TNBC patients receiving neoadjuvant chemotherapy.
3.7 Univariate and multivariate analyses of the relationships between RPL32 expression,and clinicopathological findings
In univariate analyses based upon pCR, we found a significant association between Ki-67, p53, and RPL32 expression and pCR. In multiple regression analysis, p53 (odds ratio, 0.313; p = .032) expression and RPL32 (odds ratio, 0.254; p = .026) expression were the independent factors associated with pCR (Table 2).