The Impact of Being Underweight on the Prognosis of Older Patients With Early Breast Cancer.

Background/Aim: The number of older patients with breast cancer has been increasing and a major challenge is to develop optimal treatment strategies for these patients, who often have comorbidities. Obesity is reportedly a poor prognostic factor in breast cancer, however there is limited research on underweight patients. Clarifying the relationship between physique and prognosis may contribute to the establishment of optimal treatment strategies for older patients with breast cancer. Patients and Methods: This retrospective study examined clinicopathological data from a multicenter collaborative database on 1,076 patients aged 70 years or older who had undergone curative surgery. According to the body mass index (BMI), patient physique was defined as underweight (<18.5 kg/m2), normal (18.5-24.9 kg/m2) or obese (≥25 kg/m2). In this study, we explored the relationship between the physique of patients with breast cancer and outcomes. Results: Underweight patients had a significantly lower rate of chemotherapy administration (p=0.017) and a higher rate of presence of other cancer (p=0.022). During the observation period (median of 75.2 months), 133 patients (12%) developed recurrent disease and 131 patients (12%) died. Age, BMI, tumor size, progesterone receptor and the presence of other cancer were independent factors relating to overall survival (p<0.001, p=0.027, p=0.002, p=0.008 and p=0.005, respectively). Patients with a low BMI had a significantly shorter overall survival, but there was no association with disease-free survival in this subset of patients. Conclusion: Overall survival was shorter in underweight older patients with breast cancer. Our data indicate that being underweight should be considered both in treatment decisions and in future studies of outcomes for older patients with breast cancer.

The Japanese Breast Cancer Society Clinical Practice Guidelines for systemic treatment of breast cancer, 2022 edition.

The Japanese Breast Cancer Society (JBCS) Clinical Practice Guidelines for systemic treatment of breast cancer were updated to the 2022 edition through a process started in 2018. The updated guidelines consist of 12 background questions (BQs), 33 clinical questions (CQs), and 20 future research questions (FRQs). Multiple outcomes including efficacy and safety were selected in each CQ, and then quantitative and qualitative systematic reviews were conducted to determine the strength of evidence and strength of recommendation, which was finally determined through a voting process among designated committee members. Here, we describe eight selected CQs as important updates from the previous guidelines, including novel practice-changing updates, and recommendations based on evidence that has emerged specifically from Japanese clinical trials.

E3 Ubiquitin Ligase NEDD4 Affects Estrogen Receptor α Expression and the Prognosis of Patients with Hormone Receptor-Positive Breast Cancer.

Neural precursor cell-expressed developmentally downregulated 4-1 (NEDD4) is an E3 ligase that leads to the degradation of proteins, including estrogen receptor α. We evaluated whether the expression level of NEDD4 affected the outcome of breast cancer patients. We performed a retrospective cohort study enrolling 143 patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative early breast cancer. Of the 66 patients with high NEDD4 mRNA levels (high NEDD4 group) and 77 patients with low NEDD4 mRNA levels (low NEDD4 group), 98.4% and 96.1%, respectively, of the patients had received neoadjuvant/adjuvant hormone therapy. Disease-free survival and overall survival were significantly longer in the low NEDD4 group than in the high NEDD4 group (p = 0.048 and p = 0.022, respectively). Western blotting revealed a high expression of estrogen receptor α in the NEDD4-knockdown culture cells. The proliferation of NEDD4-knockdown cells treated with tamoxifen or estradiol deprivation was suppressed, compared with that of NEDD4-expressing cells. Knockdown of NEDD4 in breast cancer cells induced the accumulation of estrogen receptor α and increased sensitivity to hormone therapy. In summary, this mechanism may lead to a better prognosis in hormone receptor-positive breast cancer patients with a low expression of NEDD4.

Successful management of pelvic recurrence of MSI-High endometrial cancer by total pelvic exenteration followed by administration of pembrolizumab:A case report.

Surgery can be curative treatment for pelvic locoregional recurrence of endometrial cancer; however, a cure is contingent on complete resection. Here, we report the case of a patient in whom recurrent endometrial tumor remained in the pelvis after resection; long-term control was achieved with postoperative administration of pembrolizumab.The patient had recurrent endometrial cancer of stage IA and was treated with chemotherapy and radiation, but tumor persisted in the pelvic cavity. We therefore attempted total pelvic exenteration, but the tumor was adherent to the pelvic wall and complete resection could not be achieved. However, postoperative administration of pembrolizumab controlled the residual tumor for more than two years without regrowth. We believe that since the resected tumor was MSI-High, the residual tumor responded well to pembrolizumab. It is not known whether cytoreductive surgery contributes to a long-term response to pembrolizumab, but at least in our patient, pembrolizumab appeared to be a very effective drug therapy for MSI-High endometrial cancer that was refractory to chemotherapy and radiotherapy.

Is adjuvant chemotherapy necessary in older patients with breast cancer?

BACKGROUND: Due to the lack of clinical trials on the efficacy of chemotherapy in older patients, an optimal treatment strategy has not been developed. We investigated whether adjuvant chemotherapy could improve the survival of older patients with breast cancer in Japan. METHODS: We retrospectively analyzed data of patients with breast cancer aged ≥ 70 years who underwent breast cancer surgery in eight hospitals between 2008 and 2013. Clinical treatment and follow-up data were obtained from the patients' medical electric records. RESULTS: A total of 1095 patients were enrolled, of which 905 were included in the initial non-matched analysis. The median age and follow-up period were 75 (range 70-93) and 6.3 years, respectively. Of these patients, 127 (14%) received adjuvant chemotherapy (Chemo group) while the remaining 778 (86%) did not (Control group). The Chemo group was younger (mean age in years 73 vs 76; P < 0.0001), had a larger pathological tumor size (mean mm 25.9 vs 19.9; P < 0.0001), and more metastatic axillary lymph nodes (mean numbers 2.7 vs 0.7; P < 0.0001) than the Control group. The disease-free survival (DFS) and overall survival (OS) did not differ significantly between the two groups (P = 0.783 and P = 0.558). After matched analyses, DFS was found to be significantly prolonged with adjuvant chemotherapy (P = 0.037); however, OS difference in the matched cohort was not statistically significant (P = 0.333). CONCLUSION: The results showed that adjuvant chemotherapy was associated with a reduced risk of recurrence, but survival benefits were limited.

PP2A-dependent TFEB activation is blocked by PIKfyve-induced mTORC1 activity.

Transcriptional factor EB (TFEB) is a master regulator of genes required for autophagy and lysosomal function. The nuclear localization of TFEB is blocked by the mechanistic target of rapamycin complex 1 (mTORC1)-dependent phosphorylation of TFEB at multiple sites including Ser-211. Here we show that inhibition of PIKfyve, which produces phosphatidylinositol 3,5-bisphosphate on endosomes and lysosomes, causes a loss of Ser-211 phosphorylation and concomitant nuclear localization of TFEB. We found that while mTORC1 activity toward S6K1, as well as other major mTORC1 substrates, is not impaired, PIKfyve inhibition specifically impedes the interaction of TFEB with mTORC1. This suggests that mTORC1 activity on TFEB is selectively inhibited due to loss of mTORC1 access to TFEB. In addition, we found that TFEB activation during inhibition of PIKfyve relies on the ability of protein phosphatase 2A (PP2A) but not calcineurin/PPP3 to dephosphorylate TFEB Ser-211. Thus when PIKfyve is inhibited, PP2A is dominant over mTORC1 for control of TFEB phosphorylation at Ser-S211. Together these findings suggest that mTORC1 and PP2A have opposing roles on TFEB via phosphorylation and dephosphorylation of Ser-211, respectively, and further that PIKfyve inhibits TFEB activity by facilitating mTORC1-dependent phosphorylation of TFEB.

A mass spectrometric method for in-depth profiling of phosphoinositide regioisomers and their disease-associated regulation.

Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide regioisomers and their low intracellular concentrations, profiling these lipids and linking a specific acyl variant to a change in biological state have been difficult. To enable the comprehensive analysis of phosphoinositide phosphorylation status and acyl chain identity, we develop PRMC-MS (Phosphoinositide Regioisomer Measurement by Chiral column chromatography and Mass Spectrometry). Using this method, we reveal a severe skewing in acyl chains in phosphoinositides in Pten-deficient prostate cancer tissues, extracellular mobilization of phosphoinositides upon expression of oncogenic PIK3CA, and a unique profile for exosomal phosphoinositides. Thus, our approach allows characterizing the dynamics of phosphoinositide acyl variants in intracellular and extracellular milieus.

Risk of immunotherapy-related narcolepsy in genetically predisposed patients: a case report of narcolepsy after administration of pembrolizumab.

BACKGROUND: Immune-related adverse events associated with immune checkpoint therapy cause autoimmune disease-like symptoms. People who carry specific genotypes or haplotypes of human leucocyte antigen (HLA) are known to be predisposed to develop autoimmune diseases including narcolepsy. Immunotherapy could be a trigger to develop narcolepsy in predisposing HLA positive patients. CASE PRESENTATION: A 66-year-old woman with stage IVB endometrial carcinosarcoma experienced daytime sleepiness and temporary muscle weakness 14 days after the administration of an immune checkpoint inhibitor, pembrolizumab. These were consistent with the main symptoms of narcolepsy with cataplexy. This patient carried a highly predisposing HLA haplotype for narcolepsy; HLA-DQB1*06:02, DRB1*15:01, DQA1*01:02 and DRB5*01:01:01. A hypocretin-1/orexin-A concentration in the patient's cerebrospinal fluid was low at 9.6 pg/mL in ELISA, and 155.5 pg/mL in radioimmunoassay that was below the normal level of 200 pg/mL. Therefore, she was diagnosed with narcolepsy tentatively according to the International Classification of Sleep Disorders, third edition diagnostic criteria for narcolepsy. The onset of narcolepsy in the 60s is very rare, and narcoleptic symptoms in our patient were likely to be caused by pembrolizumab. CONCLUSIONS: This case suggests that treatment with immune checkpoint inhibitors potentially causes narcolepsy in genetically predisposed patients.

Microsatellite instability and mismatch repair protein expressions in lymphocyte-predominant breast cancer.

The frequency of microsatellite instability (MSI) is reportedly extremely low in breast cancer, despite widespread clinical expectations that many patients would be responsive to immune-checkpoint inhibitors (ICI). Considering that some triple-negative breast cancers (TNBC) responded well to ICI in a clinical trial and that a high density of tumor-infiltrating lymphocytes (TILs) is frequently observed in other cancers with high levels of microsatellite instability (MSI-H), we hypothesized that some TNBC with a high density of TILs would be MSI-H. Medullary carcinoma (MedCa) of the breast, a rare histological type, is characterized by a high density of TILs. Considering that MedCa of the colon is often MSI-H, we suspected that MedCa in breast cancer might also include MSI-H tumors. Therefore, we conducted MSI tests on such breast cancers with a high density of TILs. The MSI status of 63 TIL-high TNBC and 38 MedCa tumors, all from Asian women who had undergone curative surgery, were determined retrospectively. DNA mismatch repair (MMR) proteins and PD-L1 expression were also investigated immunohistochemically. All samples were microsatellite stable, being negative for all microsatellite markers. TIL-high TNBC with low MLH1 protein had higher levels of PD-L1 in stromal immune cells (P = .041). MedCa tumors showed significantly higher PD-L1 expression in immune cells than in TIL-high TNBC (<.001). We found that MSI-H tumors were absent in TIL-high breast cancers. Examination of MMR proteins, not a purpose of Lynch syndrome screening, may merit further studies to yield predictive information for identifying patients who are likely to benefit from ICI.

Decreased ER dependency after acquired resistance to CDK4/6 inhibitors.

BACKGROUND: Cyclin-dependent kinase (CDK) 4/6 inhibitors represent a significant advancement in the treatment of estrogen receptor (ER)-positive human epidermal growth factor receptor 2-negative advanced breast cancer. However, mechanisms of alterations after acquired resistance to CDK4/6 inhibitors and the optimal treatment options are still not established. METHODS: Abemaciclib-resistant cell lines were established from the models of estrogen deprivation-resistant cell lines which retained ER expression and activated ER function derived from MCF-7 breast cancer cell lines. Ribocilib-resistant cell lines were established in the same method as previously reported. RESULTS: Both abemaciclib- and ribociclib-resistant cell lines showed decreased ER expression. ER transcriptional activity was maintained in these cell lines; however, the sensitivity to 4-hydroxytamoxifen and fulvestrant was almost completely lost. These cell lines did not exhibit any ERα gene mutation. Abemaciclib-resistant cell lines demonstrated low sensitivity to other CDK4/6 inhibitors; sensitivities to PI3K inhibitor, mTOR inhibitor, and chemotherapeutic drugs were maintained. CONCLUSIONS: Dependence on ER signaling appears to decrease after the development of acquired resistance to CDK4/6 inhibitors. Further, CDK4/6 inhibitor-resistant cells acquired cross-resistance to other CDK4/6 inhibitors, PI3K/Akt/mTOR inhibitor therapy and chemotherapeutic drugs might serve as optimal treatment options for such breast cancers.

Impact of circulating tumour cells on survival of eribulin-treated patients with metastatic breast cancer.

Several clinical studies have examined circulating tumour cells (CTCs). However, the application of CTCs as a predictive/prognostic marker for breast cancer patients has yet to be established, particularly the selection of suitable markers for detecting CTCs. We recently investigated CTCs, including mesenchymal status, from metastatic breast cancer patients who had received eribulin-based treatment. We found that assessment of both mesenchymal and epithelial CTCs might be important for predicting eribulin responsiveness. In the current study, we followed up the outcomes of these patients after eribulin treatment and investigated the possibility of CTC analysis results serving as prognostic markers for this patient population. Twenty-one patients were enrolled and peripheral blood samples were collected before eribulin-based treatments. CTCs were then examined using a Microfluidic Chip device. CTCs positive for vimentin and pan-cytokeratin were defined as mesenchymal and epithelial CTCs, respectively. Overall survival (OS) was assessed in relation to the number of CTCs and clinicopathological factors. During the observation period, 13 patients (62%) died due to breast cancer and the median OS was 18 months. Patients with high-grade tumours and a high total number of CTCs showed significantly shorter OS than those with low-grade tumours and smaller CTC burdens (p = 0.026 and 0.037, respectively). Patients who received eribulin as the first chemotherapy for metastatic disease showed longer OS (p = 0.006). Our data suggest that determining numbers of both mesenchymal and epithelial CTCs might predict survival for patients receiving eribulin.

SH3YL1 cooperates with ESCRT-I in the sorting and degradation of the EGF receptor.

Ubiquitinated membrane proteins such as epidermal growth factor receptor (EGFR) are delivered to early endosomes and then sorted to lysosomes via multivesicular bodies (MVBs) for degradation. The regulatory mechanism underlying formation of intralumenal vesicles en route to generation of MVBs is not fully understood. In this study, we found that SH3YL1, a phosphoinositide-binding protein, had a vesicular localization pattern overlapping with internalized EGF in endosomes in the degradative pathway. Deficiency of SH3YL1 prevents EGF trafficking from early to late endosomes and inhibits degradation of EGFR. Moreover, we show that SH3YL1 mediates EGFR sorting into MVBs in a manner dependent on its C-terminal SH3 domain, which is necessary for the interaction with an ESCRT-I component, Vps37B. Taken together, our observations reveal an indispensable role of SH3YL1 in MVB sorting and EGFR degradation mediated by ESCRT complexes.

The p21 levels have the potential to be a monitoring marker for ribociclib in breast cancer.

Although cyclin-dependent kinase (CDK) 4/6 inhibitors have exhibited remarkable results for patients with estrogen receptor (ER)-positive breast cancer in clinical trials, the mechanism of CDK4/6 inhibitor resistance remains unclear. Thus, this study aimed to investigate the mechanism of CDK4/6 inhibitor resistance using two CDK4/6 inhibitor resistant breast cancer cell lines. We established CDK6 overexpressed cell lines (MCF7-C6) from MCF-7 cells using the stably transfected CDK6 expression vector. Additionally, acquired ribociclib-resistant (RIBR) cell lines were created using ER-positive hormone-resistant cell lines by long-term exposure to ribociclib. CDK6 overexpression and the knockdown of CDK4 experiments highlight the significance of high levels of CDK4 and low levels of CDK6 in CDK4/6 inhibitor sensitivity. Moreover, RIBR cell lines did not exhibit incremental CDK6 compared with ER-positive hormone-resistant cell lines. In MCF7-C6 and RIBR cell lines, p21 levels decreased, and p21 levels were proportional to CDK4/6 inhibitor sensitivity. This study suggests that overexpression of CDK6 is one of the many possible mechanisms of resistance to CDK4/6 inhibitors. Furthermore, p21 levels have the potential to serve as a marker for CDK4/6 inhibitors independent of the resistance mechanism.

Analysis of circulating tumour cell and the epithelial mesenchymal transition (EMT) status during eribulin-based treatment in 22 patients with metastatic breast cancer: a pilot study.

BACKGROUND: Liquid biopsy approaches, such as measuring circulating tumour cells (CTCs), have recently been introduced in several clinical studies. However, the development of CTCs as a predictive marker for treatment effects on breast cancer remains an enormous task. We investigated CTCs, including epithelial mesenchymal transition (EMT) status, from metastatic breast cancer patients who had received eribulin-based treatment, which reportedly suppresses EMT as a means of tumour suppression. Our aim was to test the possibility of this method serving as a tool predicting eribulin efficacy. METHODS: Twenty-two patients were enrolled and peripheral blood samples were collected before eribulin treatment. CTCs were then examined using a Microfluidic Chip device. CTCs positive for vimentin and pan-cytokeratin were defined as mesenchymal and epithelial CTCs, respectively. Progression-free survival (PFS) and clinical response were assessable in 20 and 18 patients, respectively, in relation to the number of CTCs. RESULTS: Numbers of total CTCs were significantly increased in patients with progressive disease during treatment (p = 0.006). Median PFS was 14.6 weeks and patients with more total and mesenchymal CTCs at baseline had significantly shorter PFS (p = 0.0013 and 0.013, respectively). Multivariate logistic regression analysis revealed small number of total baseline CTCs and long disease-free survival to be related to long PFS (p = 0.0004 and 0.020, respectively). CONCLUSIONS: Our data suggest that determining both mesenchymal and epithelial CTCs at baseline might be a good tool for predicting eribulin responsiveness. Evaluation of mesenchymal CTC can be considered as a parameter in larger studies, while most clinical trials are currently employing only the detection of the epithelial cellular adhesion molecule (EpCAM).

Overcoming the Adverse Effects of Everolimus to Achieve Maximum Efficacy in the Treatment of Inoperable Breast Cancer: A Review of 11 Cases at Our Hospital.

Everolimus is an effective treatment for advanced and/or metastatic breast cancer, especially in hormone receptor-positive cases. However, adverse events have prevented considerable numbers of clinicians from using this drug. Herein, we reviewed our clinical experiences and endeavored to identify patients in whom the benefits of everolimus treatment would outweigh these adverse events. If measures were available to prevent or minimize adverse effects prior to treatment, everolimus would be a more widely applicable drug. This retrospective study involved 11 patients in whom nonresectable or recurrent breast cancers were treated with everolimus between April 2014 and January 2016. Two patients achieved a partial response (PR) and 4 showed stable disease (SD) (1 showed long SD, i.e., > 24 weeks). The response rate was 18%, and the clinical benefit rate (PR + long SD) was 27%. Regarding adverse events, interstitial pneumonia (grade 3) developed in 3 patients (18%), necessitating treatment discontinuation. When using everolimus, it may be important to select suitable patients for whom this treatment can be continued with sufficient control of adverse events. Herein, we provide information relevant to the clinical use of everolimus based on our daily practice experiences with this agent.

Predictive markers for efficacy of everolimus plus exemestane in patients with luminal HER2-negative metastatic breast cancer.

Metastatic breast cancer (MBC) is essentially incurable despite recent improvements in systemic therapies. We often encounter difficulties in choosing the most appropriate treatments, with optimal timing, for individual patients. Everolimus, one of the mTOR inhibitors, is usually used with endocrine therapy for MBC. Identification of predictive markers for everolimus-based treatment remains a major issue, but to date, no predictive markers have been established. We retrospectively investigated predictive markers for treatments with everolimus plus exemestane in patients with ER-positive and HER2-negative breast cancer. Clinicopathological features of 18 patients, with locally advanced disease or MBC given everolimus plus exemestane treatments, were examined in relation to treatment effects. Also, primary breast cancer specimens, all ER positive and HER2 negative, were immunohistochemically investigated for phospho-S6 (pS6) and PTEN, to evaluate the mTOR and PIK3CA/Akt pathways. Those showing a good clinical response had a significantly lower Ki67 labeling index than the poor responders. A similar trend was observed in pS6 level but without statistical significance. Interestingly, there was no correlation between the Ki67 labeling index and pS6, and when both indexes were low, the good clinical response rate was high. The median progression-free survival was longer in the group showing a low Ki67 labeling index (109 weeks) than in that with high Ki67 (19 weeks). There was no trend between PTEN expression and treatment effects. Our results suggest that the primary tumor in luminal HER2-negative breast cancer patients with a low Ki67 labeling index and pS6 level has the potential to respond well to everolimus plus exemestane.

Synthesis of microgel sensors for spatial and temporal monitoring of protease activity.

Proteases are involved in almost every important cellular activity, from embryonic morphogenesis to apoptosis. To study protease activity in situ, hydrogels provide a synthetic mimic of the extracellular matrix (ECM) and have utility as a platform to study activity, such as those related to cell migration, in three-dimensions. While 3-dimensional visualization of protease activity could prove quite useful to elucidate the proteolytic interaction at the interface between cells and their surrounding environment, there has been no versatile tool to visualize local proteolytic activity in real time. Here, micron-sized gels were synthesized by inverse suspension polymerization using thiolene photo-click chemistry. The size distribution was selected to avoid cellular uptake and to lower cytotoxicity, while simultaneously allowing the integration of peptide-based FRET sensors of local cell activity. Proteolytic activity of collagenase was detected within an hour via changes in fluorescence of embedded microgels; incubation of microgel sensors with A375 melanoma cells showed upregulated MMP activity in the presence of soluble fibronectins in media. The microgel sensors were readily incorporated into both gelatin and poly(ethylene glycol) (PEG) hydrogels and used to successfully detect spatiotemporal proteolytic activity of A375 melanoma cells. Finally, a tumor model was constructed from a hydrogel microwell array that was used to aggregate A375 melanoma cells, and local variations in proteolytic activity were monitored as a function of distance from the cell aggregate center.

Differences in Ki67 expressions between pre- and post-neoadjuvant chemotherapy specimens might predict early recurrence of breast cancer.

The prognosis of breast cancer patients not obtaining a pathological complete response (pCR) with neoadjuvant chemotherapy (NAC) is poorer than that of pCR patients. Identifying new prognostic factors for non-pCR patients is important because fractions of this population might benefit from novel adjuvant treatments currently under development. High Ki67 expression in remnant disease after NAC has been described as a poor prognostic factor. Studies have shown that a reduction in Ki67 expression is more often observed in good responders to chemotherapy. We hypothesized that the change in Ki67 expression might be useful for predicting patient outcomes and thus retrospectively examined pairs of biopsy and surgical specimens of breast tissue from individual patients. One hundred sixteen patients with remnant invasive disease in the breast, who received NAC and underwent surgery at our institution, were retrospectively examined. Differences in Ki67 expression between pre- and post-NAC specimens were analyzed in relation to patient outcomes. The mean Ki67 expression value after NAC was higher in patients who developed metastasis than in those without metastasis (P<.01). Tumors showing higher Ki67 expression in the surgical than in the biopsy specimen were more frequent in patients with metastasis (P<.01). This trend was more obvious in patients who developed metastasis within 1 year after surgery. Our results indicate that a difference in Ki67 expressions after versus before NAC might be an important predictor of early metastasis. Evaluating not only absolute Ki67 values, but also any changes in response to NAC, may improve the prediction of patient outcomes.

PEG-peptide hydrogels reveal differential effects of matrix microenvironmental cues on melanoma drug sensitivity.

Metastatic melanoma is highly drug resistant, though the exact mechanisms of this resistance are not completely understood. One method to study melanoma drug responsiveness in vitro is through the use of multicellular spheroids, which have been found to exhibit decreased drug sensitivity compared to traditional 2D culture on various substrates. Because it is unclear whether dimensionality, cell-matrix interactions, and/or cell-cell contacts may influence melanoma drug responsiveness, we utilized a synthetic PEG-based hydrogel to compare the responses of cells cultured on top of or encapsulated within matrices with the same adhesive ligand density, polymer density, and material properties. We found that depending on the stage of progression at which the melanoma cells were derived, the cells responded differently to PLX4032 treatment, a commercially available melanoma drug. In particular, early stage WM35 cells were insensitive to dimensionality (i.e., 2D versus 3D culture), while metastatic A375 cells exhibited decreased responsiveness in 3D compared to 2D. To further understand the role of the microenvironment in early stage melanoma cells, we tested single WM35 cells and multicellular WM35 spheroids in 3D. The results revealed that the spheroids were similarly sensitive to PLX4032 treatment compared to single cell encapsulations. Collectively, this study implicates the role that 3D microenvironments (i.e., dimensionality) may play in observed melanoma drug responsiveness, and the potential lack of influence of cell-matrix interactions over cell-cell contacts in early stages of melanoma resistance to PLX4032-induced apoptosis.

Regulation of CD44 expression and focal adhesion by Golgi phosphatidylinositol 4-phosphate in breast cancer.

CD44, a transmembrane receptor, is expressed in the standard or variant form and plays a critical role in tumor progression and metastasis. This protein regulates cell adhesion and migration in breast cancer cells. We previously reported that phosphatidylinositol-4-phosphate (PI(4)P) at the Golgi regulates cell migration and invasion in breast cancer cell lines. In this study, we showed that an increase in PI(4)P levels at the Golgi by knockdown of PI(4)P phosphatase SAC1 increased the expression of standard CD44, variant CD44, and ezrin/radixin phosphorylation and enhanced the formation of focal adhesions mediated by CD44 and ezrin/radixin in MCF7 and SK-BR-3 cells. In contrast, knockdown of PI 4-kinase IIIß in highly invasive MDA-MB-231 cells decreased these factors. These results suggest that SAC1 expression and PI(4)P at the Golgi are important in tumor progression and metastasis and are potential prognostic markers of breast cancers.