According to the latest Global Cancer Statistics Report, breast cancer in women surpassed lung cancer to become the world's largest cancer, with approximately 2.3 million new cases in 2020, accounting for 11.7% of all new cancer cases [1].
Triple-negative breast cancer (TNBC) refers to breast cancer in which estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2 are all negative, accounting for 10%-20% of invasive breast cancers. Compared with other subtypes, TNBC has the characteristics of strong invasiveness, easy transfer, and poor prognosis, because of its lack of targeted receptors and limited treatment options, making it a clinical problem that needs to be overcome urgently [2,3].
Platinum-based agents have been shown to show good efficacy in neoadjuvant chemotherapy and metastatic TNBC therapy [4]. However, platinum drug chemotherapy has high economic cost and greater side effects, and some patients are insensitive to platinum chemotherapy, which delays the treatment time. Therefore, it is of clinical importance to explore effective predictors of treatment response and screen the TNBC subgroup that may benefit.
In recent years, nucleolar small RNAs (snoRNAs) have received widespread attention, and studies have shown that snoRNAs are stable in peripheral plasma and serum, easy to enrich and detect, closely related to the clinical pathological features and prognosis of tumors, and can be used as potential biomarkers for liquid biopsy [5]. This undoubtedly brings new research ideas and hope to researchers.
Recently, it was found that SNORD33 is a marker for predicting the efficacy of platinum drugs, and they also revealed the molecular mechanism of SNORD33 regulating platinum resistance. The research results were published online in the prestigious journal Molecular Cancer.
▲ Screenshot of the first page of the paper
Let's take a look at how this study is conducted.
First, the researchers constructed the cisplatin-resistant TNBC cell line MDA-MB-231/DDP, which is 5 times more resistant to cisplatin than the maternal cell line MDA-MB-231. A total of 277 RNAs were found to be abnormally expressed in MDA-MB-231/DDP cells by RNA sequencing, with snoRNAs with the highest rate of variability (12.3%). Of all the SNORNAs, SNORD33 had the largest variation and the most significant decrease.
Subsequently, after silencing expression of SNORD33 in MDA-MB-231, MDA-MB-468, and SUM149PT cells, the researchers conducted cell experiments to explore the effects of SNORD33 on TNBC cell function.
The results showed that after SNORD33 knockout, the proliferation ability of breast cancer cells was enhanced, and the apoptosis cells were significantly reduced. They also detected a significant increase in levels of anti-apoptotic proteins (Mcl-1 and Bcl-2) and decreased expression of pro-apoptotic proteins (activated caspase-3 and caspase-9) in SNORD33 knockout cells.
The above studies showed that SNORD33 expression was significantly reduced in cisplatin-resistant TNBC cells, and down-regulated SNORD33 expression enhanced the resistance of TNBC cells to cisplatin.
▲ Effect of SNORD33 on biological function of TNBC cells
Increased resistance to cisplatin in 231/DDP cells (Figure a); heat map analysis showed differences in snoRNAs expression in 231/DDP cells compared to 231 cells (Figure b), and volcano analysis showed that the expression of SNORD33 was most significantly reduced (Figure c); downregulation of SNORD33 expression in 231/DDP cells (Figure d); increased cisplatin resistance in TNBC cells after SNORD33 knockout (Figure e).
In in vitro cell function experiments, the effect of SNORD33 on cisplatin resistance in TNBC cells is obvious, so what is the expression of SNORD33 in the plasma of TNBC patients and the relationship between it and the prognosis of patients?
First, the researchers collected plasma from 255 patients with mTNBC from three clinical trials, including 45 platinum-free patients and 209 platinum-containing agents in the first-line chemotherapy regimen (81 in the training cohort, 128 in the validation cohort, and 114 in the combined cohort of 209 randomly tested for BRCA gene mutations).
In the training cohort, 81 samples were divided into SNORD33 high-expression group and SNORD33 low-expression group according to the SNORD33 expression level in plasma, and the baseline characteristics between the two groups were basically flat. The analysis found that the progression-free survival (PFS) of the low SNORD33 group was significantly shorter than that of the high SNORD33 group (6.6 months vs 10.1 months).
The analysis results in the validation queue and the combined queue were consistent with the above conclusions: the PFS of the low SNORD33 group was significantly reduced (validation queue: 6.5vs10.1 months; combined queue: 6.6vs10.1 months).
In the combined cohort, the overall survival (OS) in the low SNORD33 group was significantly reduced (22.2 months versus 40.6 months) compared with the high SNORD33 group. Univariate Cox regression analysis showed that low SNORD33 expression, number of metastases, visceral metastases, and liver metastases were significantly associated with decreased PFS and OS. In multivariate analysis, SNORD33 is an independent predictor of platinum-based chemotherapy PFS.
At the same time, in the three study cohorts, plasma SNORD33 was significantly higher in patients who achieved clinical efficacy (CB) than those who did not achieve CB.
In addition, changes in plasma SNORD33 levels in patients receiving non-platinum chemotherapy were not associated with PFS.
The above findings show that the predicted value of SNORD33 expression level is platinum-specific. The most studied state of the platinum-sensitive biomarker BRCA mutation in breast cancer is not related to plasma SNORD33 levels and PFS in platinum chemotherapy patients.
ROC analysis found that plasma levels of SNORD33 as a predictor of platinum chemotherapy outcomes had better clinical application value (AUC = 0.652) compared with other single clinical pathopathological risk factors such as liver metastases and the number of metastases.
Finally, based on the plasma SNORD33 levels, liver metastases and the number of metastases in the multivariate Cox regression model, the researchers plotted a linear array that predicted the timing of PFS in platinum-containing regimens, which was used to predict the likelihood of clinical remission at different times of platinum treatment in patients, and the information provided by prognostic stromat can provide important reference value for clinicians when choosing a regimen.
▲ The relationship between the expression level of SNORD33 in the plasma of patients with mTNBC and the corresponding prognosis
Patient selection and study design (figure f); relationship between median progression-free survival (PFS) and serum expression levels of SNORD33 in plasma in mTNBC patients treated with platinum-containing regimens, training cohort (Figure g), validation cohort (Fig.h); median progression-free survival (PFS) in mTNBC patients treated with non-platinum regimens versus expression levels of SNORD33 in plasma (Fig.i); In patients who achieved clinical efficacy, Plasma SNORD33 levels were significantly elevated (Figure j); BRCA mutations were not associated with plasma SNORD33 levels (Figure k) and were also not associated with PFS (Figure l); plasma SNORD33 had the highest predictive value for PFS compared to the presence or absence of liver metastases and the number of metastases (Figure m); the probability of developing PFS in patients with TNBC after the start of platinum therapy (Figure n); the calibration curve of the line plot (Figure o).
Next, the researchers further explored the mechanism by which SNORD33 affects platinum resistance.
First, the researchers used an RNA pull-down test to bind to mass spectrometry and found that SNORD33 regulates the expression of the MeCP2-targeted gene by preventing the binding of methylated binding protein 2 (MeCP2) to methylated CpG DNA. Downregulation of SNORD33 expression can release the transcriptional inhibition activity of MeCP2, thereby inhibiting the expression of pro-apoptotic genes, reducing apoptosis in tumor cells, and increasing the resistance of triple-negative breast cancer cells to platinum.
▲ In MDA-MB-231 cells, RPL13A, DICER enzyme, or SNORD32a are knocked out (Figure a); RPL13A, DICER enzyme, or SNORD32a gene knockout does not alter cell resistance to platinum (Figure b); Silver stained bands show experimental and control bands (Figure c); RNA pull-down and RIP (RNA immunoprecipitation) analysis in MDA-MB-231 cells (Figure d) Knockout of the SNORD33 gene in MDA-MB-231 cells does not alter MeCP2 mRNA and protein levels (Figure e); in MDA-MB-231 cells knocked out of SNORD33, mRNA levels of the MeCP2 target gene are reduced. (Figure f); SNORD33 gene knockout promotes the binding of MeCP2 to downstream target gene promoters (Figure g); SNORD33 gene knockout does not alter the co-inhibition factors mSIN3A and HDAC1 binding to MeCP2 (Figure i).
Subsequently, in order to further confirm the above conclusions, the researchers found that after knocking out MeCP2 in the TNBC cell line with SNORD33 knockout, meCP2 downregulation weakened the reduction of pro-apoptotic proteins and apoptosis induced by SNORD33 gene knockout, which increased the expression of anti-apoptotic proteins, and partially reversed platinum resistance in TNBC cells due to SNORD33 deletion.
▲ MeCP2 downregulation partially reverses SNORD33 knockout induced platinum resistance (Figure j); Flow Cytometry determination of cisplatin-treated apoptosis (Figure k); Westernblot detection of changes in apoptosis-related proteins in cisplatin-treated cells (Figure l)
The above data show that SNORD33 regulates the expression of apoptosis-related genes by influencing the binding of MeCP2 to the target protein, and thus affects the apoptosis of cells, and MeCP2 is considered a potential target for SNORD33 to regulate platinum resistance in TNBC cells.
Overall, in this study, the researchers first successfully constructed platinum-resistant TNBC cell lines, detected abnormal expression of RNA through RNA sequencing, and found small nucleolar RNA, SNORD33; and through a series of studies, confirmed that SNORD33 was closely related to TNBC cell resistance to cisplatin, and then further explored the expression of plasma SNORD33 in mTNBC patients and its relationship with patient prognosis. A column plot to predict the PFS time of the platinum-containing scheme is constructed. Finally, they also revealed the molecular mechanism by which SNORD33 affects platinum resistance. The whole process is interlocked, and this study provides a highly efficient, simple, and reliable plasma marker for the prediction of clinical outcomes in patients with TNBC and the choice of clinical treatment regimen.
We are also looking forward to the clinical study of SNORD33 in neoadjuvant chemotherapy and other platinum-containing treatment options for neoadjuvant chemotherapy and other tumor patients, hoping to see that SNORD33 can be applied to clinical treatment as soon as possible and bring more benefits to tumor patients."
bibliography:
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