[Frontiers in Bioscience 3, d944-960, September 1, 1998]
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CAVEAT LECTOR




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BIOLOGICAL AND MOLECULAR BASIS OF HUMAN BREAST CANCER

Jose Russo, Xiaoqi Yang, Yun-Fu Hu, Betsy A. Bove, Yajue Huang, Ismael D.C.G. Silva, Quivo Tahin, Yuli Wu, Nadia Higgy, Abdel Zekri, and Irma H. Russo

Breast Cancer Research Laboratory, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA

Received 12/17/97 Accepted 7/21/98

4. FACTORS INFLUENCING SUSCEPTIBILITY OF HBEC TO CELL TRANSFORMATION

4.1. Lobular differentiation

Using our established in vitro system that allows us to efficiently culture HBEC whose growth characteristics in vitro reflect the in vivo degree of lobular development and the rate of cell proliferation (6), we tested on HBEC the transforming potential of chemical carcinogens known to be of etiological importance in various experimental models of mammary cancer (29). Our results indicate that HBEC of Lob 1, obtained from young nulliparous women, which are less differentiated, are more proliferative and more susceptible to be transformed by chemical carcinogens than those of the more differentiated Lob 3 from older and parous women (6). These results indicate that the susceptibility of HBEC in vitro is influenced by differentiation status of the breast in vivo. Whereas we do not have a complete elucidation of the genes involved in the process of mammary gland differentiation, we have recently cloned a gene, namely, mammary-derived growth inhibitor (MDGI), from the primary culture of HBEC (30). Using in situ hybridization techniques, we have demonstrated that the expression of MDGI is absent in the least differentiated Lob 1 and 2, low in the moderately differentiated Lob 3 and maximal in the most differentiated Lob 4 (30) (Table 1). In consideration of other observations that the MDGI locus, mapped to chromosome 1p21-23 (31), is frequently lost in breast tumors (32) and that MDGI expression is associated with tumor suppression in breast cancer cells (33), it is reasonable to speculate that MDGI might be a tumor suppressor and silenced in the very early stage of carcinogenesis. Overexpression of MDGI in the otherwise susceptible HBEC (e.g., MCF-10F cells) would confer resistance to chemical carcinogenesis in these cells. Experiments are currently in progress to test this hypothesis.

Table 1. Expression of MDGI in relation to lobular differentiation in human breast

Case #

Agea

Lobule Typeb

MDGIc

7

46

1

+/-

8

48

1

+/-

10

39

1

+/-

6

49

1/2

+

9

47

1/2

+/-

2

36

2

+

5

41

2/3

+

1

43

3

++

3

29

4

++++

4

35

4

++++

aAge of the donor at the time of reduction mammoplasty surgery.

bLobules type 1, 2, 3, and 4, classified according to histopathogical criteria described previously (11).

cIn situ hybridization with the antisense riboprobe of human MDGI. The intensity of the hybridization signal was scored as negative (-), weakly positive (+), moderately positive (++) and strongly positive (+++/++++).

4.2. Genetic predisposition

The primary cultures obtained from women with no family history of breast cancer, when treated with the carcinogens in vitro, exhibit an increase in survival efficiency (11, 13), which is perceived to precede the acquisition of anchorage independence (34, 35). In contrast, treatment of HBEC from women with family history of breast cancer with the same carcinogens induces the formation of colonies in agar-methocel (14) (Figure 2). Colonies formed from the treated cells showed considerable anchorage-independent growth during the 21-day assay period (Figure 3). However, when individual colonies were isolated, they failed to grow. Since formation of colonies in agar is generally construed as indicating anchorage-independent growth, a hallmark of neoplastic cells, our results clearly showed that HBEC from women with familial history of breast cancer manifested phenotypic changes indicative of initial stages of neoplastic transformation in response to the carcinogen treatment. Since inherited and acquired (spontaneous and induced) genetic changes can predispose an individual to both premalignant and malignant transformation of a specific organ (36, 37), it is reasonable to speculate that genetic predisposition in women with familial history of breast cancer may confer inherited susceptibility to environmental chemical carcinogens.

Figure 2. Survival and colony efficiencies in agar methocel of chemical carcinogen-treated HBEC in vitro (Reproduced with permission from Ref. 11).

Figure 3. Colonies formed in agar methocel in chemical carcinogen-treated HBEC from women with familial history of breast cancer (Reproduced with permission from Ref. 11).

4.3. Cell immortalization

Since in vitro treatment of HBEC in primary cultures with chemical carcinogens failed to induce the full expression of malignant transformation, we decided to use the protocols developed for primary culture of HBEC with the immortalized cell line MCF-10F in order to elucidate whether immortalization is required for the expression of the full malignant phenotype. Upon treatment of MCF-10F cells with 7, 12-dimethylbenzo[a ]anthracene (DMBA), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (NMU), or benzo[a ]pyrene (BP), the treated cells showed altered morphology and altered pattern of growth, increased growth rate and anchorage independent growth in agar-methocel after 8-10 passages (around 157 days after the treatment). Control cells never developed colonies, whereas carcinogen-treated cells formed colonies from which the following clones were derived: D1, D2, and D3, from DMBA, M4 from MNNG, and BP1, BP2, BP5, BP6, BP7, and BP10 from BP treated cells (38) (Figure 4). All clones grew at a faster rate than their respective parental cells. Based upon this growth advantage, selected cell populations of clones BP1 and BP2 were isolated at approximately 446 days post-treatment; they were named BP1E and BP2B, respectively. BP1 and BP1E cells showed an increase in anchorage-independent growth, chemotaxis and invasiveness. From the D3 clone the D3-1 cell line was originated. It showed increased chemotactic and invasive capabilities, but to a lesser degree than BP1E. The tumorigenic potential of these cells was tested by inoculation into SCID mice. After 105 days after injection, mammary tumor developed from the BP1E cell line (Figure 4). None of the other BP clones or those derived from DMBA or MNNG-treated cells formed tumors (Table 2).

Figure 4. Diagrammatic representation of the evolution of MCF-10F cells treated with the chemical carcinogens.

Table 2. Expression of transformed phenotypes in MCF-10F and its derivatives1

Phenotypes

#130

MCF-10F

BP1

BP1E

In vitro Growth

  • Doubling time (hr)

N/A

93

80

44

Anchorage-Independent Growth

  • Survival Efficiency (%)

N/A

91.9 35.0

N/A

N/A

  • Colony Efficiency (%)

N/A

0

45 12

88 10

  • Day 7 Colony Size (mm2)

N/A

0

167 26

687 28

Duct-like Formation in Collagen Gel

N/A

+

+/-

-

Invasiveness Index in Matrigel

N/A

130 7

457 43

820 181

Tumorigenesis in SCID Mice

  • No. Cells Injected (x 106)

N/A

7 - 30

20

10

  • No. Animals w/ Tumors/Total No.

N/A

0/5

0/8

13/13

  • Average Tumor Size (mm3)

N/A

0

0

9.5

  • Latency Period (days)

N/A

?

?

101

Notes: 1. This table is modified from ref. 38.

These results led us to conclude that chemical carcinogens induce the expression of the definitive neoplastic phenotype, tumorigenesis, as long as HBEC are immortalized prior to carcinogen exposure. This model allowed us to isolate clone of cells expressing different stages of progression to neoplastic transformation, which will certainly facilitate our studies on the molecular mechanisms of cell transformation. Specifically, our in vitro model will allow us to answer the following important questions: 1. Which is the molecular mechanism of cell immortalization? 2. Which are the genetic changes involved in the process of cell transformation? 3. Which are the genetic changes related to the process of tumorigenesis? 4. Which of the genes identified in the process of tumorigenesis are important to revert the process? and 5. Which of the genetic changes are relevant to the process of neoplastic development in vivo?