FOXA1 (HMV4726)

The FOXA1 (Forkhead Box A1) gene, located at chromosome 14q21.1 codes for a critical “pioneer factor” protein. It binds to nucleosomal DNA, opens local chromatin, and recruits or stabilizes other transcription factors, several of which are hormone receptors or related proteins such as for example estrogen receptor, androgen receptor or GATA3. FOXA1 thereby establishes lineage-specific transcriptional programs in endoderm-derived organs (liver, lung, pancreas, prostate, mammary gland) and in luminal mammary epithelial cells. FOXA1 is essential for the development of several organs including pancreas, liver, prostate, and the lung. FOXA1 knockout mice survive embryogenesis but die postnatally with hypoglycaemia – due to a lack of glucagon – and other metabolic defects. FOXA1 germline mutations are not known to occur in humans. In cancer, FOXA1 shows context‑dependent roles. Somatic FOXA1 mutations, focal amplifications, and overexpression are recurrent in prostate, breast, lung, bladder and other cancers. The functional and clinical consequences of these alterations are not fully understood. Depending on the tumor type, both reduced and increased expression has been found to be related to aggressive cancer phenotype.

Images describing the EGFRvIII staining pattern in normal tissues obtained by the antibody HMV4726 are shown in our “Normal Tissue Gallery”.

In normal tissues, FOXA1 staining is most intense in prostatic epithelium, urothelium, respiratory epithelium, and in a subset of breast epithelial cells. FOXA1 positivity also occurs in hepatocytes, intestinal epithelium, salivary glands, gallbladder epithelium, pneumocytes, fallopian tube, sebaceous cells of the skin, and in non-keratinizing squamous epithelium.  These findings are largely consistent with the RNA data described in the Human Protein Atlas (Tissue expression FOXA1). 

Positive control = Colon: A strong nuclear FOXA1 staining should be seen in crypt cells the mucosa. The FOXA1 staining intensity should gradually decrease towards the surface epithelium.

Negative control = Kidney: FOXA1 staining must not be seen in any cell type of the kidney.

Normal tissue gallery

Cancer tissue gallery

IHC users have different preferences on how the stains should look like. Some prefer high staining intensity of the target stain and even accept some background. Others favor absolute specificity and lighter target stains. Factors that invariably lead to more intense staining include higher concentration of the antibody and visualization tools, longer incubation time, higher temperature during incubation, higher temperature and longer duration of the heat induced epitope retrieval (slide pretreatment). The impact of the pH during slide pretreatment has variable effects and depends on the antibody and the target protein.

All images and data shown here and in our image galleries are obtained by the manual protocol described below. Other protocols resulting in equivalent staining are described as well.

Manual protocol

Freshly cut sections should be used (less than 10 days between cutting and staining). Heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7,8 Target Retrieval Solution buffer. Apply HMV4726 at a dilution of 1:150 at 37°C for 60 minutes. Visualization of bound antibody by the EnVision Kit (Dako, Agilent) according to the manufacturer’s directions.

• What is the prevalence and clinical significance of aberrant FOXA1 expression in different cancer types?
• Can FOXA1 IHC be used to distinguish clinically relevant tumor types? 
• How do specific FOXA1 mutations mechanistically reprogram chromatin to drive therapy resistance in prostate and breast cancer? 
• Which tumor contexts show FOXA1 as driver versus tumor suppressor, and what determines that switch?

There are two ways how the specificity of antibodies can be documented for immunohistochemistry on formalin fixed tissues. These are: 1. Comparison with a second independent method for target expression measurement across a large number of different tissue types (orthogonal strategy), and 2. Comparison with one or several independent antibodies for the same target and showing that all positive staining results are also seen with other antibodies for the same target (independent antibody strategy). 

Orthogonal validation: For the antibody HMV4726 specificity is strongly supported by a comparison of its staining pattern with FOXA1 RNA expression data in normal tissues which were collected in three independent RNA screening studies, including the Human Protein Atlas (HPA) RNA-seq tissue dataset, the FANTOM5 project, and the Genotype-Tissue Expression (GTEx) project, and which are summarized in the Human Protein Atlas (Tissue expression FOXA1). In line with existing RNA data, strong/significant FOXA1 staining was highest in prostatic epithelium, urothelium, a subset of breast epithelial cells, hepatocytes, stomach glands, and it was also seen in intestinal epithelium, salivary glands, gallbladder, pneumocytes, fallopian tube, and in squamous epithelium. In line with absence of FOXA1 RNA, FOX1 immunostaining by HMV4726 was not seen in endometrium, placenta, ovary, muscle, testis, epididymis, seminal vesicle, kidney, pancreas, all endocrine tissues, and the brain.

Comparison of antibodies: True expression of FOXA1 in all cells with FOXA1 positivity by HMV4726 is corroborated by a confirmation of each individual staining seen by HMV4726 in 76 different normal tissue types by a commercially available independent second antibody (termed “validation antibody”). An additional nuclear staining of the reference antibody in several other epithelial tissues such as the endometrium, the pancreas, glia cells of the brain, corpus luteum of the ovary, endocervix, and chief cells of the epididymis was considered an antibody specific cross-reactivity of the validation antibody as it was not seen by HMV4726.

Normal tissue gallery