Human mammary cancer progression model recapitulates methylation events associated with breast premalignancy

Introduction

We have previously identified a rare subpopulation of variant human mammary epithelial cells (vHMEC) with repressed p16^INK4A that exist in disease-free women yet display premalignant properties, suggesting that they have engaged the process of malignant transformation. In order to gain insight into the molecular alterations required for vHMEC to progress to malignancy, and to characterize the epigenetic events associated with early progression, we examined the effect of oncogenic stress on the behavior of these cells.

Methods

HMEC that express p16^INK4A and vHMEC that do not, were transduced with constitutively active Ha-rasV12 and subsequently exposed to serum to determine whether signals from the cellular microenvironment could cooperate with ras to promote the malignant transformation of vHMEC. Epigenetic alterations were assessed using methylation-specific polymerase chain reaction (PCR).

Results

vHMEC expressing Ha-rasV12 (vHMEC-ras) bypassed the classic proliferative arrest that has been previously documented in normal fibroblasts following oncogenic stress, and that we also observe here in normal HMEC. Moreover, vHMEC-ras cells exhibited many additional alterations that are observed during progression to malignancy such as the generation of chromosomal abnormalities, upregulation of telomerase activity, immortalization following exposure to serum, and anchorage-independent growth, but they did not form tumors following orthotopic injection in vivo. Associated with their early progression to malignancy was an increase in the number of genes methylated, two of which (RASSF1A and SFRP1) were also methylated in other immortalized mammary cell lines as well as in breast cancer cells and tissues.

Conclusions

We have characterized a mammary progression model that recapitulates molecular and methylation alterations observed in many breast cancers. Our data suggest that concomitant methylation of RASSF1A and SFRP1 marks an early event in mammary transformation and may thus have prognostic potential.     

Analysis of <Emphasis Type="Italic">CUL-5</Emphasis> expression in breast epithelial cells,breast cancer cell lines,normal tissues and tumor tissues

Background  

The chromosomal location of CUL-5 (11q 22-23) is associated with LOH in breast cancer, suggesting that CUL-5 may be a tumor suppressor. The purpose of this research was to determine if there is differential expression of CUL-5 in breast epithelial cells versus breast cancer cell lines, and normal human tissues versus human tumors. The expression of CUL-5 in breast epithelial cells (HMEC, MCF-10A), and breast cancer cells (MCF-7, MDA-MB-231) was examined using RT-PCR, Northern blot analysis, and Western blot analysis. The expression of mRNA for other CUL family members (CUL-1, -2, -3, -4A, and -4B) in these cells was evaluated by RT-PCR. A normal human tissue expression array and a cancer profiling array were used to examine CUL-5 expression in normal human tissues and matched normal tissues versus tumor tissues, respectively.     

Characterization of human breast cancer epithelial cells (HBCEC) derived from long term cultured biopsies

Introduction

For a more individualized therapeutic approach we explored a protease-free method to culture primary cells from breast cancer biopsies.

Methods and Results

Tumor tissue from breast cancer patients after surgery was cultured ex vivo without enzymatic digestion for more than one year and revealed the continuous outgrowth of adherent and proliferating primary cell populations. Immunofluorescence staining of these human breast cancer-derived epithelial cells (HBCEC) and quantification by flow cytometry revealed nearly exclusively cytokeratin-expressing cells. Analysis of surface markers during long term tumor culture of primary HBCEC (more than 476d) demonstrated a prominent expression of CD24, CD44 and MUC1 (CD227). According to aging markers, expression of senescence-associated β-galactosidase revealed little if any positive staining in a primary tumor-derived HBCEC population after 722d in culture, whereas the majority of normal human mammary epithelial cells (HMEC) demonstrated senescent cells already after a culture period of 32d. In this context, HBCEC populations derived from a tumor culture after 152d and 308d, respectively, exhibited a significant telomerase activity, suggesting continuous proliferative capacity. Treatment with several chemotherapeutic compounds and their combinations revealed distinct cytotoxic effects among HBCEC from different breast cancer patients, indicating an individualized response of these tumor-derived primary cells.

Conclusion

The protease-free outgrowth of primary HBCEC offers a patient-specific approach to optimize an individually-designed cancer therapy. Moreover, HBCEC from long term breast tumor tissue cultures resemble tumor cell-like properties by an intact ECM formation and a stable cell surface protein expression providing a reproducible screening platform to identify new biomarkers and to test new therapeutics in individual tumor samples.     

Y-box-binding protein 1 (YB1) in breast carcinomas: Relation to aggressive tumor phenotype and identification of patients at high risk for relapse

Aims

To investigate the expression pattern of Y-box-binding protein 1 (YB1) in breast carcinomas, its clinicopathological and prognostic value, and its association with the breast cancer stem cell phenotype [CD44⁺/CD24^−/low].

Methods and results

Immunohistochemistry was performed on 225 paraffin embedded specimens of invasive breast carcinomas to detect the expression of the proteins YB1, ER, PR, HER2, p53, Ki67, bcl-2, CD44 and CD24. YB1 protein was detected in the nuclei, the cytoplasm and the stroma of the tumor cells. Cytoplasmic YB1 was detected more often in carcinomas of ductal type (p = 0.002), of higher nuclear grade (p < 0.001), with lack of ER expression (p = 0.002), positive expression of p53 and Ki67 (p = 0.002 and p = 022, respectively), and with present CD44⁺/CD24^−/low breast cancer stem cells (p = 0.001), while its association with bcl-2 was found to be inverse (p = 0.042). Nuclear YB1 was found to exert unfavorable impact on the disease-free survival of the unselected patients (p = 0.05) and the patients having been subjected to adjuvant chemotherapy and radiotherapy (p = 0.036 and p = 0.05, respectively).

Conclusions

Cytoplasmic YB1 is associated with an aggressive and “stem cell-like” tumor phenotype, while nuclear localization discriminates patients at high risk for recurrence, especially those who are subjected to chemo- and radiotherapy.     

Overexpression of peroxiredoxin I and thioredoxin1 in human breast carcinoma

Introduction

For a more individualized therapeutic approach we explored a protease-free method to culture primary cells from breast cancer biopsies.

Methods and Results

Tumor tissue from breast cancer patients after surgery was cultured ex vivo without enzymatic digestion for more than one year and revealed the continuous outgrowth of adherent and proliferating primary cell populations. Immunofluorescence staining of these human breast cancer-derived epithelial cells (HBCEC) and quantification by flow cytometry revealed nearly exclusively cytokeratin-expressing cells. Analysis of surface markers during long term tumor culture of primary HBCEC (more than 476d) demonstrated a prominent expression of CD24, CD44 and MUC1 (CD227). According to aging markers, expression of senescence-associated β-galactosidase revealed little if any positive staining in a primary tumor-derived HBCEC population after 722d in culture, whereas the majority of normal human mammary epithelial cells (HMEC) demonstrated senescent cells already after a culture period of 32d. In this context, HBCEC populations derived from a tumor culture after 152d and 308d, respectively, exhibited a significant telomerase activity, suggesting continuous proliferative capacity. Treatment with several chemotherapeutic compounds and their combinations revealed distinct cytotoxic effects among HBCEC from different breast cancer patients, indicating an individualized response of these tumor-derived primary cells.

Conclusion

The protease-free outgrowth of primary HBCEC offers a patient-specific approach to optimize an individually-designed cancer therapy. Moreover, HBCEC from long term breast tumor tissue cultures resemble tumor cell-like properties by an intact ECM formation and a stable cell surface protein expression providing a reproducible screening platform to identify new biomarkers and to test new therapeutics in individual tumor samples.     

The mechanisms by which polyamines accelerate tumor spread

Background

The pathogenic role of beta-HPVs in non melanoma skin cancer (NMSC), is not still completely understood, and literature data indicate that they might be at least cofactors in the development of certain cutaneous squamous cell carcinomas. However, only few reports contain data on basal cell carcinoma (BCC). The HPVs interact with many cellular proteins altering their function or the expression levels, like the p16^INK4a and Akt. Our study aimed to determine the presence of different beta -HPV types and the expression of p16^INK4a and Akt in BCC, the commonest NMSC, in the normal appearing perilesional skin and in forehead swab of 37 immunocompetent patients.

Methods

The expression of p16^INK4a and Akt, by immunohistochemistry, and the HPV DNA, by nested PCR, were investigated in each sample.

Results

No correspondence of HPV types between BCC and swab samples was found, whereas a correspondence between perilesional skin and BCC was ascertained in the 16,7% of the patients. In BCC, 16 different types of beta HPV were found and the most frequent types were HPV107 (15,4%), HPV100 (11,5%) and HPV15 (11,5%) all belonging to the beta HPV species 2. Immunohistochemistry detected significant p16^INK4a expression in almost all tumor samples (94,3%) with the highest percentages (> 30%) of positive cells detected in 8 cases. A statistically significant (p = 0,012) increase of beta HPV presence was detected in p16^INK4a strongly positive samples, in particular of species 2. pAkt expression was detected in all tumor samples with only 2 cases showing rare positive cells, whereas Akt2 expression was found in 14 out of 35 BCC (40%); in particular in HPV positive samples over-expressing p16^INK4a.

Conclusions

Our data show that p16^INK4a and pAkt are over-expressed in BCC and that the high expression of p16^INK4a and of Akt2 isoform is often associated with the presence of beta-HPV species 2 (i.e. HPV 15). The association of these viruses with the up-regulation of p16^INK4a and Akt/PI3K pathway suggests that in a subtype of BCC these viruses may exert a role in the carcinogenesis or in other, still undefined, biological property of these tumors. If this particular type of BCC reflects a different biology it will remain undisclosed until further studies on a larger number of samples will be performed.     

<Emphasis Type="Italic">CDKN1C</Emphasis>/p57<Superscript>kip2</Superscript>is a candidate tumor suppressor gene in human breast cancer

Background  

CDKN1C (also known as p57^KIP2) is a cyclin-dependent kinase inhibitor previously implicated in several types of human cancer. Its family members (CDKN1A/p21^CIP1 and B/p27^KIP1) have been implicated in breast cancer, but information about CDKN1C's role is limited. We hypothesized that decreased CDKN1C may be involved in human breast carcinogenesis in vivo.     

Targeting tumour energy metabolism potentiates the cytotoxicity of 5-aminolevulinic acid photodynamic therapy

Background:

Cancerous cells usually exhibit increased aerobic glycolysis, compared with normal tissue (the Warburg effect), making this pathway an attractive therapeutic target.

Methods:

Cell viability, cell number, clonogenic assay, reactive oxygen (ROS), ATP, and apoptosis were assayed in MCF-7 tumour cells and corresponding primary human mammary epithelial cells (HMEC).

Results:

Combining the glycolysis inhibitors 2-deoxyglucose (2DG; 180 mM) or lonidamine (300 μM) with 10 J cm⁻² 5-aminolevulinic acid (ALA) photodynamic therapy (PDT) increases MCF-7 cytotoxicity (by 3.5-fold to 70% death after 24 h, and by 10-fold in 9-day clonogenic assays). However, glycolysis inhibition only slightly increases HMEC PDT cytotoxicity (between two-fold and three-fold to a maximum of 9% death after 24 h). The potentiation of PDT cytotoxicity only occurred if the glycolysis inhibitors were added after ALA incubation, as they inhibited intracellular accumulation of photosensitiser if coincubated with ALA.

Conclusion:

As 2DG and lonidamine are already used as cancer chemotherapeutic agents, our results are directly translatable to combination therapies with existing topical PDT.     

MTAP is an independent prognosis marker and the concordant loss of MTAP and p16 expression predicts short survival in non-small cell lung cancer patients

Objectives

Methylthioadenosine phosphorylase (MTAP), a ubiquitously expressed protein, plays important roles in purine biosynthesis. Locating near to each other on chromosome 9p21-22, codeletion of the MTAP and p16^Ink4A genes have been reported in non-small cell lung cancer (NSCLC). The aim of this study is to determine the respective prognostic value of MTAP and p16 by considering their correlation in NSCLC patients.

Materials and methods

We analyzed MTAP and p16 protein expression by immunohistochemical staining on 99 NSCLC tissue microarray samples. The association between MTAP and p16 expression levels and prognosis were analyzed using the Kaplan–Meier method and Cox proportional hazards model for prognosis.

Results

Patients with a low MTAP expression level had poor overall survival (P = 0.010) and disease-free survival (P = 0.002). Low p16 expression indicated a trend toward poor overall survival (P = 0.138) and disease-free survival (P = 0.199). There was a significant positive correlation between MTAP and p16 expression levels (Spearman's ρ = 0.402, P < 0.001). By multivariate analyses, the MTAP expression level retained its independent prognostic power and p16 expression loss of the correlation with prognosis. Concordant loss of MTAP and p16 expression was observed in 24 out of 99 patients (24.2%). Patients with concordant loss of MTAP and p16 expression had the worst prognosis compared to patients with high expression of both markers.

Conclusion

MTAP expression is an independent prognostic factor and has greater prognostic significance than p16 expression in NSCLC. Concordant loss of MTAP and p16 expression indicates poor outcomes in lung cancer patients.     

Increasing CD44+/CD24- tumor stem cells, and upregulation of COX-2 and HDAC6, as major functions of HER2 in breast tumorigenesis

Background  

Cancer cells are believed to arise primarily from stem cells. CD44⁺/CD24^- have been identified as markers for human breast cancer stem cells. Although, HER2 is a well known breast cancer oncogene, the mechanisms of action of this gene are not completely understood. Previously, we have derived immortal (M13SV1), weakly tumorigenic (M13SV1R2) and highly tumorigenic (M13SV1R2N1) cell lines from a breast epithelial cell type with stem cell phenotypes after successive SV40 large T-antigen transfection, X-ray irradiation and ectopic expression of HER2/C-erbB2/neu. Recently, we found that M13SV1R2 cells became non-tumorigenic after growing in a growth factor/hormone-deprived medium (R2d cells).     

The in situ local immune response,tumour senescence and proliferation in colorectal cancer

Background:

Immune cell infiltrates are important determinants of colorectal cancer (CRC) outcome. Their presence may be driven by tumour or host-specific factors. From previous studies in mice, senescence, a state of cell cycle arrest, may moderate tumour progression through upregulation of antitumour immune responses. The relationships between senescence and immune infiltrates have not previously been studied in humans. We explore whether a marker of senescence (p16^ink4a) in combination with low level expression of a proliferation marker (ki-67) relate to T cell infiltrates in CRC, and whether p16^ink4a, Ki-67 and immune infiltrates have similar prognostic value.

Methods:

Immunostaining of p16^inka and Ki-67 was performed within a CRC tissue microarray. Nuclear p16^inka and Ki-67 were categorised as high/low. T-cell markers, CD3, CD45RO, CD8 and FOXP3 were scored separately as high/low grade in three areas of the tumour: the invasive margin (IM), tumour stroma and cancer cell nests (CCNs).

Results:

Two hundred and thirty stage I–III cancers were studied. High nuclear p16^ink4a was expressed in 63% and high proliferation (Ki-67 >15%) in 61%. p16^ink4a expression was associated with reduced CD45RO+ cells at the IM (P<0.05) and within the stroma (P<0.05) and reduced CD8+ cells at the IM (P<0.01). A low Ki-67 proliferative index was associated with reduced density of CD3+ cells in CCNs (P<0.01), reduced CD45RO+ cells at the IM (P<0.05) and within the CCNs (P<0.001), reduced FOXP3+ cells at the IM (P<0.001), within the stroma (P=0.001) and within CCNs (P<0.001) and reduced CD8+ cells at the IM (P<0.05) and within the CCNs (P<0.05). Tumours with both a low proliferative index and expression of p16^ink4a demonstrated similar consistent relationships with reduced densities of T-cell infiltrates. On multivariate analysis, TNM stage (P<0.001), low CD3 cells at the IM (P=0.014), low CD8 cells at the IM (P=0.037), low proliferation (Ki-67; P=0.013) and low senescence (p16^ink4a; P=0.002) were independently associated with poorer cancer survival.

Conclusion:

Senescence, proliferation and immune cell infiltrates are independent prognostic factors in CRC. Although related to survival, p16^ink4a-associated senescence is not associated with an upregulation of antitumour T-cell responses.     

Prediction of non-sentinel lymph node metastasis in early breast cancer by assessing total tumoral load in the sentinel lymph node by molecular assay

Introduction

The one-step nucleic acid amplification (OSNA) is a molecular procedure that yields a semiquantitative result for detection of nodal metastasis. Size of metastasis in the sentinel lymph node (SLN) by conventional histology has been described as a predictive factor for additional axillary metastasis. The objective of this study is to quantify intraoperatively the total tumoral load (TTL) in the positive SLNs assessed by OSNA and to determine whether this TTL predicts non-SLN metastasis in patients with clinically node negative early stage breast cancer.

Methods

306 patients with cT1-3N0 invasive breast cancer who had undergone intraoperative SLN evaluation by OSNA were included. TTL was defined as the addition of CK19 mRNA copies of each positive SLN (copies/μL).

Results

TTL was a predictive factor of additional non-SLN metastasis in the complete axillary lymph node dissection (cALND) (OR, 1.67; 95% CI, 1.18–2.35). In the multivariate analysis, the TTL was a predictor of non-SLN metastasis in HR positive patients (OR, 1.69; 95% CI, 1.19–2.41). In our cohort of patients, with a TTL ≤1.2 × 10⁵ copies/μL, there was a specificity of 85.3% and negative predictive value (NPV) of 80%. If we consider only the HR positive patients, with a TTL ≤5 × 10⁵ copies/μL there was a specificity of 86.7% and NPV of 83.7%.

Conclusions

TTL assessed by OSNA assay predicts for additional non-SLN metastasis and this intraoperative tool can help guiding decisions on performing a cALND in breast cancer patients.     

Adherence to the 2018 World Cancer Research Fund/American Institute for Cancer Research Cancer Prevention Recommendations and cancer risk: A systematic review and meta-analysis

Background

The World Cancer Research Fund/American Institute for Cancer Research Cancer Prevention Recommendations are lifestyle-based guidelines that aim to reduce cancer risk. A systematic review and meta-analysis of studies investigating associations between a score for adherence to the 2018 Cancer Prevention Recommendations and cancer risk was conducted.

Methods

MEDLINE, Embase, Web of Science, and Scopus were searched for studies published to November 28, 2022. In meta-analysis, the estimated risk ratios and 95% CIs for adherence score as a continuous (per 1-point increment) and categorical (highest vs. lowest score category) variable using random-effects models were estimated.

Results

Eighteen studies (11 cohort; seven case-control) were included investigating incidence of breast (n = 7), colorectal (n = 5), prostate (n = 2), lung (n = 2), pancreatic (n = 1), endometrial (n = 1), unknown primary cancer (n = 1), chronic lymphocytic leukemia (n = 1), and overall (any) cancer (n = 1). The summary risk ratio per 1-point increment in adherence score was 0.89 (95% CI, 0.85–0.93; I² = 76.5%; n = 7) for breast cancer, 0.88 (95% CI, 0.84–0.91; I² = 26.2%; n = 4) for colorectal cancer, and 0.92 (95% CI, 0.86–0.98, I² = 66.0%; n = 2) for lung cancer. There were no significant associations with prostate or other cancers. Meta-analysis results using categorical adherence score variables were consistent with these findings.

Conclusions

Greater adherence to the 2018 World Cancer Research Fund/American Institute for Cancer Research Cancer Prevention Recommendations was associated with lower risk of breast, colorectal, and lung cancers. Future studies investigating associations with risk of other forms of cancer are warranted.

PROSPERO registration number

CRD42022313327.     

The CD44<Superscript>+</Superscript>/CD24<Superscript>-</Superscript>phenotype is enriched in basal-like breast tumors

Introduction  

Human breast tumors are heterogeneous and consist of phenotypically diverse cells. Breast cancer cells with a CD44⁺/CD24^- phenotype have been suggested to have tumor-initiating properties with stem cell-like and invasive features, although it is unclear whether their presence within a tumor has clinical implications. There is also a large heterogeneity between tumors, illustrated by reproducible stratification into various subtypes based on gene expression profiles or histopathological features. We have explored the prevalence of cells with different CD44/CD24 phenotypes within breast cancer subtypes.     

Triage of women with ASCUS and LSIL cytology

BACKGROUND.

The identification of a small percentage of high‐grade cervical intraepithelial neoplasias (HGCIN) among patients with minor cytological abnormalities (atypical squamous cells of undetermined significance [ASCUS] and/or low‐grade squamous intraepithelial lesions [LSIL] group) is a major problem in cytology‐based cervical cancer screening. The authors investigated the efficacy of p16^INK4a as a biomarker to identify samples of patients with HGCIN among those with an ASCUS or LSIL result in Papanicolaou cytology.

METHODS.

Consecutive liquid‐based cytology specimens of 137 ASCUS and 88 LSIL results were selected from gynecologists who adopted a triage regimen with biopsy under colposcopy 2 months later, independent of the p16^INK4a result. p16^INK4a stained slides were prepared and independently read by 2 observers, who used a recently described score to categorize p16^INK4a stained squamous cells. The endpoint of the study was detection of a biopsy‐confirmed HGCIN.

RESULTS.

The overall sensitivity and specificity of p16^INK4a positive cells with a nuclear score >2 for diagnosis of HGCIN in ASCUS and LSIL cases combined was 96% and 83%, respectively. The sensitivity and specificity in the ASCUS group was 95% and 84%, and 100% and 81% in the LSIL group, respectively. Two observers had a high concordance in assessing p16^INK4a stained cells (κ value of 0.841).

CONCLUSIONS.

These data suggested that the use of p16^INK4a as a biomarker combined with nuclear scoring of p16^INK4a positive cells in cervical cytology to triage ASCUS and/or LSIL cases allows identification of HGCIN with good sensitivity and specificity. Cancer (Cancer Cytopathol) 2007. © 2006 American Cancer Society.     

Epigenetic control of the basal-like gene expression profile via Interleukin-6 in breast cancer cells

Background  

Basal-like carcinoma are aggressive breast cancers that frequently carry p53 inactivating mutations, lack estrogen receptor-α (ERα) and express the cancer stem cell markers CD133 and CD44. These tumors also over-express Interleukin 6 (IL-6), a pro-inflammatory cytokine that stimulates the growth of breast cancer stem/progenitor cells.     

Quantitative copy number analysis by Multiplex Ligation-dependent Probe Amplification (MLPA) of BRCA1-associated breast cancer regions identifies BRCAness

Introduction  

Our group has previously employed array Comparative Genomic Hybridization (aCGH) to assess the genomic patterns of BRCA1-mutated breast cancers. We have shown that the so-called BRCA1-like^aCGH profile is also present in about half of all triple-negative sporadic breast cancers and is predictive for benefit from intensified alkylating chemotherapy. As aCGH is a rather complex method, we translated the BRCA1^aCGH profile to a Multiplex Ligation-dependent Probe Amplification (MLPA) assay, to identify both BRCA1-mutated breast cancers and sporadic cases with a BRCA1-like^aCGH profile.