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More than 70% of breast cancers are estrogen receptor-positive (ER+). Endocrine therapy that blocks estrogen signaling remains the cornerstone of treatment, yet relapses continue to affect many patients. Cyclin-dependent kinases 4 and 6 (CDK4/6) regulate the G1-S phase transition in the cell cycle, and pharmacological inhibition of this pathway has been successfully leveraged to reduce recurrence. CDK4/6 inhibitors combined with endocrine therapy are now the standard of care, although determining the optimal patient population for treatment remains a key challenge. A newly published study provides important insight, showing that loss of the NF1/neurofibromin tumor suppressor confers greater sensitivity to CDK4/6 inhibition, as these tumors rely heavily on CDK4/6 activity for survival under endocrine therapy.

Background Ductal carcinoma in situ (DCIS) is the most common type of in situ premalignant breast cancers. What drives DCIS to invasive breast cancer is unclear. Basal-like invasive breast cancers are aggressive. We have previously shown that NRAS is highly expressed selectively in basal-like subtypes of invasive breast cancers and can promote their growth and progression. In this study, we investigated whether NRAS expression at the DCIS stage can control transition from luminal DCIS to basal-like invasive breast cancers. Methods Wilcoxon rank-sum test was performed to assess expression of NRAS in DCIS compared to invasive breast tumors in patients. NRAS mRNA levels were also determined by fluorescence in situ hybridization in patient tumor microarrays (TMAs) with concurrent normal, DCIS, and invasive breast cancer, and association of NRAS mRNA levels with DCIS and invasive breast cancer was assessed by paired Wilcoxon signed-rank test. Pearson’s correlation was calculated between NRAS mRNA levels and basal biomarkers in the TMAs, as well as in patient datasets. RNA-seq data were generated in cell lines, and unsupervised hierarchical clustering was performed after combining with RNA-seq data from a previously published patient cohort. Results Invasive breast cancers showed higher NRAS mRNA levels compared to DCIS samples. These NRAS high lesions were also enriched with basal-like features, such as basal gene expression signatures, lower ER, and higher p53 protein and Ki67 levels. We have shown previously that NRAS drives aggressive features in DCIS-like and basal-like SUM102PT cells. Here, we found that NRAS -silencing induced a shift to a luminal gene expression pattern. Conversely, NRAS overexpression in the luminal DCIS SUM225 cells induced a basal-like gene expression pattern, as well as an epithelial-to-mesenchymal transition signature. Furthermore, these cells formed disorganized mammospheres containing cell masses with an apparent reduction in adhesion. Conclusions These data suggest that elevated NRAS levels in DCIS are not only a marker but can also control the emergence of basal-like features leading to more aggressive tumor activity, thus supporting the therapeutic hypothesis that targeting NRAS and/or downstream pathways may block disease progression for a subset of DCIS patients with high NRAS .

Ras GTPases are powerful drivers for tumorigenesis, but directly targeting Ras for treating cancer remains challenging. The growth and transforming activity of the aggressive basal-like breast cancer (BLBC) are driven by N-Ras. To target N-Ras in BLBC, this study screened existing pharmacologically active compounds for the new ability to induce N-Ras degradation, which led to the identification of flunarizine (FLN), previously approved for treating migraine and epilepsy. The FLN-induced N-Ras degradation was not affected by a 26S-proteasome inhibitor. Rather, it was blocked by autophagy inhibitors. Furthermore, N-Ras can be seen co-localized with active autophagosomes upon FLN treatment, suggesting that FLN alters the autophagy pathway to degrade N-Ras. Importantly, FLN treatment recapitulated the effect of N-RAS silencing in vitro by selectively inhibiting the growth of BLBC cells, but not that of breast cancer cells of other subtypes. In addition, in vivo FLN inhibited tumor growth of a BLBC xenograft model. In conclusion, this proof-of-principle study presents evidence that the autophagy pathway can be coerced by small molecule inhibitors, such as FLN, to degrade Ras as a strategy to treat cancer. FLN has low toxicity and should be further investigated to enrich the toolbox of cancer therapeutics.

Background The INT6 gene was first discovered as a site of integration in mouse mammary tumors by the mouse mammary tumor virus; however, INT6 ’s role in the development of human breast cancer remains largely unknown. By gene silencing, we have previously shown that repressing INT6 promotes transforming activity in untransformed human mammary epithelial cells. In the present study, guided by microarray data of human tumors, we have discovered a role of Int6 in stromal fibroblasts. Results We searched microarray databases of human tumors to assess Int6’s role in breast cancer. While INT6 expression levels, as expected, were lower in breast tumors than in adjacent normal breast tissue samples, INT6 expression levels were also substantially lower in tumor stroma. By immunohistochemistry, we determined that the low levels of INT6 mRNA observed in the microarray databases most likely occurs in stromal fibroblasts, because far fewer fibroblasts in the tumor tissue showed detectable levels of the Int6 protein. To directly investigate the effects of Int6 repression on fibroblasts, we silenced INT6 expression in immortalized human mammary fibroblasts (HMFs). When these INT6 -repressed HMFs were co-cultured with breast cancer cells, the abilities of the latter to form colonies in soft agar and to invade were enhanced. We analyzed INT6 -repressed HMFs and found an increase in the levels of a key carcinoma-associated fibroblast (CAF) marker, smooth muscle actin. Furthermore, like CAFs, these INT6 -repressed HMFs secreted more stromal cell–derived factor 1 (SDF-1), and the addition of an SDF-1 antagonist attenuated the INT6 -repressed HMFs’ ability to enhance soft agar colony formation when co-cultured with cancer cells. These INT6 -repressed HMFs also expressed high levels of mesenchymal markers such as vimentin and N-cadherin. Intriguingly, when mesenchymal stem cells (MSCs) were induced to form CAFs, Int6 levels were reduced. Conclusion These data suggest that besides enhancing transforming activity in epithelial cells, INT6 repression can also induce fibroblasts, and possibly MSCs as well, via mesenchymal-mesenchymal transitions to promote the formation of CAFs, leading to a proinvasive microenvironment for tumorigenesis.

Over half of breast cancer cases are estrogen-dependent and strategies to combat estrogen-dependent breast cancer have been to either block the activation of estrogen receptor (ER) or diminish the supply of estrogens. Our previous work documented that estrogen-independent expression of progesterone receptor (PR) in MCF-7 cells markedly disrupted the effects of estrogen. In this study, we have developed an adenovirus-mediated gene delivery system to study the specific involvement of PR isoform A (PR-A) and PR-B in the anti-estrogenic effect and its mechanism of action. The results revealed that PR-B, but not PR-A, exhibited distinct anti-estrogenic effect on E2-induced cell growth, gene expression, and ER-ERE interaction in a ligand-independent manner. The anti-estrogenic effect of PR-B was also associated with heightened metabolism and increased cellular uptake of estradiol-17β (E2). We have also found that the B-upstream segment of PR-B alone was able to inhibit E2-induced ER-ERE interaction and cellular uptake of E2. Although PR-A alone did not affect E2-induced ER activity, it antagonized the anti-estrogenic effect of PR-B in a concentration-dependent manner. The findings suggest an important mechanism of maintaining a favorable level of ER activity by PR-A and PR-B in estrogen target cells for optimal growth and differentiation. The potential anti-estrogenic mechanism of PR-B may be exploited for breast cancer therapy.

While Ras is well-known to function on the plasma membrane (PM) to mediate growth factor signaling, increasing evidence suggests that Ras has complex roles in the cytoplasm. To uncover these roles, we screened a cDNA library and isolated H-Ras-binding proteins that also influence Ras functions. Many isolated proteins regulate trafficking involving endosomes; CHMP6/VPS20 and VPS4A, which interact with ESCRT-III (Endosomal Sorting Complex Required for Transport-III), were chosen for further study. We showed that the binding is direct and occurs in endosomes. Furthermore, the binding is most efficient when H-Ras has a functional effector-binding loop, and is GTP-bound and ubiquitylated. CHMP6 and VPS4A also bound to N-Ras but not K-Ras. Repressing CHMP6 and VPS4A blocked Ras-induced transformation, which correlated with inefficient Ras localization to the PM as measured by cell fractionation and photobleaching. Moreover, silencing CHMP6 and VPS4A also blocked epidermal growth factor receptor (EGFR) recycling. These data suggest that Ras interacts with key ESCRT-III components to promote recycling of itself and EGFR back to the PM to create a positive feedback loop to enhance growth factor signaling.

Mitochondria have a crucial role in regulating energy metabolism and their dysfunction has been linked to tumorigenesis. Cancer diagnosis and intervention have a great interest in the development of new agents that target biomolecules within mitochondria. However, monitoring and modulating mitochondria RNA (mtRNA), an essential component in mitochondria, in cells is challenging due to limited functional research and the absence of targeting agents. In this study, we designed and synthesized a fluorescent quinolinium derivative, QUCO-1, which actively lit up with mtRNA in both normal and cancer cells in vitro. Additionally, we evaluated the function of QUCO-1 as an mtRNA ligand and found that it effectively induced severe mitochondrial dysfunction and OXPHOS inhibition in RKO colorectal cancer cells. Treatment with QUCO-1 resulted in apoptosis, cell cycle blockage at the G2/M phase, and the effective inhibition of cell proliferation. Our findings suggest that QUCO-1 has great potential as a promising probe and therapeutic agent for mtRNA, with the potential for treating colorectal cancer.

A predictive model utilizing near-infrared spectroscopy was developed to estimate the loss on drying, total contents of crocin I and crocin II, and picrocrocin content of saffron. Initially, the LD values were determined using a moisture-ash analyzer, while HPLC was employed for measuring the total contents of crocin I, crocin II, and picrocrocin. The near-infrared spectra of 928 saffron samples were collected and preprocessed using first derivative, standard normal variable transformation, detrended correction, multivariate scattering correction, Savitzky–Golay smoothing, and mean centering methods. Leveraging the partial least squares method, regression models were constructed, with parameters optimized through a selective combination of the above six preprocessing methods. Subsequently, prediction models for loss on drying, total contents of crocin I and crocin II, and picrocrocin content were established, and the prediction accuracy of the models was verified. The correlation coefficients and root mean square error of loss on drying, total contents of crocin I and crocin II, and picrocrocin content demonstrated high accuracy, with R2 values of 0.8627, 0.8851, and 0.8592 and root mean square error values of 0.0260, 0.0682, and 0.0465. This near-infrared prediction model established in the present study offers a precise and efficient means of assessing loss on drying, total contents of crocin I and crocin II, and picrocrocin content in saffron and is useful for the development of a rapid quality evaluation system.

Remimazolam, a novel ultra-short-acting benzodiazepine, is a potential sedative for non-general anesthesia surgery in the elderly. This study aimed to investigate the appropriate sedative dosage of remimazolam and its effects on perioperative cognitive function in elderly patients undergoing non-general anesthesia surgery. This multicenter, placebo-controlled trial enrolled 330 elderly patients undergoing non-general anesthesia procedures at eight centers in China from July 2021 to February 2022, with 238 ultimately completing the study. The primary endpoints were the dose of successful sedation with remimazolam and the changes in perioperative cognitive function. Adverse events were recorded to assess drug safety. The induction dose of remimazolam for sedation in spinal anesthesia in elderly patients was 5.38 mg (95% confidence interval [CI], 5.20-5.56), maintained at a rate of 0.223 mg·kg ·h (95% CI, 0.201-0.237) with no serious adverse effects. Compared with the standard saline group, there was no statistical difference in the MMSE scores on Day 2 morning (P = 0.886), Day 2 afternoon (P = 0.864), and Day 7 (P = 0.613), and no statistical difference in the MoCA scores on Day 2 morning (P = 0.687), Day 2 afternoon (P = 0.827), and Day 7 (P = 0.483) in remimazolam group. Remimazolam besylate is an effective sedative for elderly patients undergoing neuraxial anesthesia. It was successfully induced at a dose of 5.38 mg and maintained at 0.223 mg·kg ·h , demonstrating a good safety profile without affecting short-term postoperative cognitive function. http://www.chictr.org.cn (ChiCTR2100048744).

Cullin-RING ubiquitin ligase 4 (CRL4) is closely correlated with the incidence and progression of ovarian cancer. DDB1- and CUL4-associated factor 13 (DCAF13), a substrate-recognition protein in the CRL4 E3 ubiquitin ligase complex, is involved in the occurrence and development of ovarian cancer. However, its precise function and the underlying molecular mechanism in this disease remain unclear. In this study, we confirmed that DCAF13 is highly expressed in human ovarian cancer and its expression is negatively correlated with the overall survival rate of patients with ovarian cancer. We then used CRISPR/Cas9 to knockout DCAF13 and found that its deletion significantly inhibited the proliferation, colony formation, and migration of human ovarian cancer cells. In addition, DCAF13 deficiency inhibited tumor proliferation in nude mice. Mechanistically, CRL4-DCAF13 targeted Fraser extracellular matrix complex subunit 1 (FRAS1) for polyubiquitination and proteasomal degradation. FRAS1 influenced the proliferation and migration of ovarian cancer cell through induction of the focal adhesion kinase (FAK) signaling pathway. These findings collectively show that DCAF13 is an important oncogene that promotes tumorigenesis in ovarian cancer cells by mediating FRAS1/FAK signaling. Our findings provide a foundation for the development of targeted therapeutics for ovarian cancer.